Compositions of, and methods for, alpha-1 anti trypsin Fc fusion molecules

ABSTRACT

A novel method of treating and preventing bacterial diseases is provided. In particular, the present invention relates to compositions and methods for inhibition of Gram negative, Gram positive and acid fast bacilli in general and tuberculosis (TB),  Mycobacterium avium  complex (MAC), and anthrax in particular. Thus, the invention relates to modulation of cellular activities, including macrophage activity, and the like. More particularly, the present invention relates to the inhibitory compounds comprising naturally occurring and man-made inhibitors of serine protease.

PRIORITY

This application is a divisional application of U.S. patent applicationSer. No. 14/465,733, now allowed, which is a continuation application ofU.S. patent application Ser. No. 13/958,443, filed on Aug. 2, 2013, nowU.S. Pat. No. 9,499,606, which is a continuation application of U.S.patent application Ser. No. 13/297,095, filed Nov. 15, 2011, now U.S.Pat. No. 8,633,305, which is a continuation application of U.S. patentapplication Ser. No. 12/555,895, filed Sep. 9, 2009, now abandoned,which is a divisional application of U.S. patent application Ser. No.10/926,051, filed Aug. 26, 2004, now U.S. Pat. No. 7,850,970, whichclaims priority to U.S. Provisional Application No. 60/497,703, filedAug. 26, 2003. These applications are incorporated herein by referencein their entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for inhibitionof bacterial infections comprising Gram negative, Gram positive, andacid fast bacilli in general and Mycobacterium tuberculosis (TB),Mycobacterium avium complex (MAC), and anthrax in particular, as well asto therapeutic treatment of diseases or disorders that involve infectionof macrophages. Thus, the invention relates to modulation of cellularactivities, including macrophage activity, inhibition of toxin, and thelike. More particularly, the present invention also relates toinhibitory compounds comprising naturally occurring and man-made serineprotease inhibitors and antagonists.

BACKGROUND OF THE INVENTION

Serine Proteases

Serine proteases serve an important role in human physiology bymediating the activation of vital functions. In addition to their normalphysiological function, serine proteases have been implicated in anumber of pathological conditions in humans. Serine proteases arecharacterized by a catalytic triad consisting of aspartic acid,histidine and serine at the active site.

The naturally occurring serine protease inhibitors are usually, but notalways, polypeptides and proteins which have been classified intofamilies primarily on the basis of the disulfide bonding pattern and thesequence homology of the reactive site. Serine protease inhibitors,including the group known as serpins, have been found in microbes, inthe tissues and fluids of plants, animals, insects and other organisms.Protease inhibitor activities were first discovered in human plasma byFermi and Pemossi in 1894. At least nine separate, well-characterizedproteins are now identified, which share the ability to inhibit theactivity of various proteases. Several of the inhibitors have beengrouped together, namely α1-antitrypsin-proteinase inhibitor,antithrombin III, antichymotrypsin, C1-inhibitor, and α2-antiplasmin,which are directed against various serine proteases, i.e., leukocyteelastase, thrombin, cathepsin G, chymotrypsin, plasminogen activators,and plasmin. These inhibitors are members of theα1-antitrypsin-proteinase inhibitor class. The protein α2-macroglobulininhibits members of all four catalytic classes: serine, cysteine,aspartic, and metalloproteases. However, other types of proteaseinhibitors are class specific. For example, theα1-antitrypsin-proteinase inhibitor (also known as (α1-antitrypsin orAAT) and inter-α-trypsin inhibitor inhibit only serine proteases,α1-cysteine protease inhibitor inhibits cysteine proteases, andα1-anticollagenase inhibits collagenolytic enzymes of the metalloenzymeclass.

Human neutrophil elastase (NE) is a proteolytic enzyme secreted bypolymorphonuclear leukocytes in response to a variety of inflammatorystimuli. The degradative capacity of NE, under normal circumstances, ismodulated by relatively high plasma concentrations of α1-antitrypsin.However, stimulated neutrophils produce a burst of active oxygenmetabolites, some of which (hypochlorous acid for example) are capableof oxidizing a critical methionine residue in α1-antitrypsin. Oxidizedα1-antitrypsin has been shown to have a limited potency as a NEinhibitor and it has been proposed that alteration of thisprotease/antiprotease balance permits NE to perform its degradativefunctions in localized and controlled environments.

α1-antitrypsin is a glycoprotein of MW 51,000 with 417 amino acids and 3oligosaccharide side chains. Human α1-antitrypsin was named anti-trypsinbecause of its initially discovered ability to inactivate pancreatictrypsin. Human α1-antitrypsin is a single polypeptide chain with nointernal disulfide bonds and only a single cysteine residue normallyintermolecularly disulfide-linked to either cysteine or glutathione. Thereactive site of α1-antitrypsin contains a methionine residue, which islabile to oxidation upon exposure to tobacco smoke or other oxidizingpollutants. Such oxidation reduces the biological activity ofα1-antitrypsin; therefore substitution of another amino acid at thatposition, i.e. alanine, valine, glycine, phenylalanine, arginine orlysine, produces a form of α1-antitrypsin which is more stable.α1-antitrypsin can be represented by the following amino acid sequence,SEQ ID NO. 63:

1         01         01         01         01        0MPSSVSWGIL LAGLCCLVPV SLAEDPQGDA AQKTDTSHHD QDHPTFNKITPNLAEFAFSL YRQLAHQSNS TNIFFSPVSI ATAFANLSLG TKADTHDEIL 100EGLNFNLTEI PEAQIHEGFQ ELLRTLNQPD SQLQLTTGNG LFLSEGLKLVDKFLEDVKKL YHSEAFTVNF GDHEEAKKQI NDYVEKGTQG KIVDLVKELD 200RDTVFALVNY IFFKGKWERP FEVKDTEDED HVDQVTTVK VPMMKRLGMFNIQHCKKLSS WVLLMKYLGN ATAIFFLPDE GKLQHLENEL THDIITKFLE 300NEDRRSASLH LPKLSITGTY DLKSVLGQLG ITKVFSNGAD LSGVTEEAPLKLSKAVHKAV LTIDEKGTEA AGAMFLEAIP MSIPPEVKFN KPFVFLMIEQ 400NTKSPLFMGK VVNPTQK 417Ciliberto, et al. in Cell 1985, 41, 531-540. The critical amino acidsequence near the carboxyterminal end of α1-antitrypsin is shown in boldand underlined and is pertinent to this invention (details of thesequence can be found for example in U.S. Pat. No. 5,470,970 asincorporated by reference).

The normal plasma concentration of AAT ranges from 1.3 to 3.5 mg/mlalthough it can behave as an acute phase reactant and increases 3-4-foldduring host response to inflammation and/or tissue injury such as withpregnancy, acute infection, and tumors. It easily diffuses into tissuespaces and forms a 1:1 complex with a target protease, principallyneutrophil elastase. Other enzymes such as trypsin, chymotrypsin,cathepsin G, plasmin, thrombin, tissue kallikrein, and factor Xa canalso serve as substrates. The enzyme/inhibitor complex is then removedfrom circulation by binding to serpin-enzyme complex (SEC) receptor andcatabolized by the liver and spleen. Humans with circulating levels ofα1-antitrypsin less than 15% of normal are susceptible to thedevelopment of lung disease, e.g., familial emphysema, at an early age.Familial emphysema is associated with low ratios of α1-antitrypsin toserine proteases, particularly elastase. Therefore, it appears that thisinhibitor represents an important part of the defense mechanism againstattack by serine proteases.

α1-antitrypsin is one of few naturally occurring mammalian serineprotease inhibitors currently approved for the clinical therapy ofprotease imbalance. Therapeutic α1-antitrypsin has been commerciallyavailable since the mid 80s and is prepared by various purificationmethods (see for example Bollen et al., U.S. Pat. No. 4,629,567;Thompson et al., U.S. Pat. Nos. 4,760,130; 5,616,693; WO 98/56821).Prolastin® is a trademark for a purified variant of α1-antitrypsin andis currently sold by Bayer Company (U.S. Pat. No. 5,610,285 Lebing etal., Mar. 11, 1997). Recombinant unmodified and mutant variants ofα1-antitrypsin produced by genetic engineering methods are also known(U.S. Pat. No. 4,711,848); methods of use are also known, e.g.,(α1-antitrypsin gene therapy/delivery (U.S. Pat. No. 5,399,346 to FrenchAnderson et al.).

The two known cellular mechanisms of action of serine proteases are bydirect degradative effects and by activation of G-protein-coupledproteinase-activated receptors (PARs). The PAR is activated by thebinding of the protease followed by hydrolysis of specific peptidebonds, with the result that the new N-terminal sequences stimulate thereceptor. The consequences of PAR activation depend on the PAR type thatis stimulated and on the cell or tissue affected and may includeactivation of phospholipase C beta (PLCβ), activation of protein kinaseC (PKC) and inhibition of adenylate kinase (ADK) (Dery, O. and Bunnett,N. W. Biochem Soc Trans 1999, 27, 246-254; Altieri, D. C. J. Leukoc Biol1995, 58, 120-127; Dery, O. et al. Am J. Physiol 1998, 274,C1429-C1452).

TB and MAC

Mycobacterium is a genus of bacteria which are aerobic, mostly slowgrowing, slightly curved or straight rods, sometimes branching andfilamentous, and distinguished by acid-fast staining. Typically,mycobacteria are gram-positive obligate aerobes. The genus Mycobacteriumincludes the highly pathogenic organisms that cause tuberculosis (M.tuberculosis and sometimes M. bovis) and leprosy (M. leprae). There are,however, many other species of Mycobacterium such as M.avium-intracellulare, M. chelonei (also known as borstelense andabscessus), M. africanum, M. marinium (also known as balnei andplatypoecilus), M. buruli (also known as ulcerans), M. fortuitum (alsoknown as giae, minetti, and ranae), M. haemophilum, M. intracellulare,M. kansasii (also known as luciflavum), M. littorale (also known asxenopi), M. malmoense, M. marianum (also known as scrofulaceum andparaffinicum), M. simiae, M. szulgai, and M. ulcerans.

Mycobacteria which are pathogenic for animals but not believed to bepathogenic for humans include the following: M. avium-intracellulare(also known as brunense), M. flavascens, M. lepraemurium, M. microti,and M. paratuberculosis (which is the causative agent for Johne'sDisease, and perhaps Crohn's disease). The following species of thegenus Mycobacterium are believed to be non-pathogenic: M. gordonae (alsoknown as aquae), M. gastri, M. phlei (also known as moelleri and astimothy bacillus), M. nonchromogenicum, M. smegmatis, M. terrae, M.triviale, and M. vaccae.

Additionally, certain mycobacteria other than M. tuberculosis and M.bovis are alternatively known as non-tuberculosis mycobacteria. They aredivided into four groups, also known as Runyon groups, based onpigmentation and growth rate. Each group includes several species. GroupI refers to slow-growing photochromogens; Group II refers toslow-growing scotochromogens; Group III refers to slow-growingnonphotochromogens; and Group IV refers to rapidly-growing mycobacteria.The non-tuberculosis mycobacteria are also called atypical or anonymousmycobacteria.

Tuberculosis is an acute or chronic infectious disease caused byinfection with M. tuberculosis. Tuberculosis is a major disease indeveloping countries, as well as an increasing problem in developedareas of the world, with approximately 8 million new cases and 3 milliondeaths each year (See Styblo et al., Bull. Int. Union Tuberc. 56:118-125(1981). Although the infection may be asymptomatic for a considerableperiod of time, the disease is most commonly manifested as an acuteinflammation of the lungs, resulting in fever and a nonproductive cough.If left untreated, serious complications and death typically result.

Although it is known that tuberculosis can generally be controlled usingextended antibiotic therapy, such treatment is not sufficient to preventthe spread of the disease. Infected individuals may be asymptomatic, butcontagious, for some time. In addition, although compliance with thespecific treatment regimen is critical, patient behavior is oftendifficult to monitor. Treatment regimens often require six to twelvemonths of uninterrupted therapy. As a result, some patients do notcomplete the course of treatment, thus leading to ineffective treatmentand development of antibiotic resistance. Effective vaccination andaccurate, early diagnosis of the disease are needed in order to inhibitthe spread of tuberculosis. Vaccination with live bacteria remains themost efficient method for inducing protective immunity. The most commonMycobacterium employed in the live vaccine is Bacillus Calmette-Guerin(BCG), an avirulent strain of Mycobacterium bovis. Some countries, suchas the United States, however, do not vaccinate the general publicbecause of concerns regarding the safety and efficacy of BCG.

M. tuberculosis is an intracellular pathogen that infects macrophagesand is able to survive within the harsh environment of the phagolysosomein macrophages. Most inhaled bacilli are destroyed by activated alveolarmacrophages. However, the surviving bacilli multiply in macrophages andare released upon cell death, which signals the infiltration oflymphocytes, monocytes and macrophages to the site. Antigenicstimulation of T cells requires presentation by MHC molecules. Lysis ofthe bacilli-laden macrophages is mediated by the delayed-typehypersensitivity (DTH) cell-mediated immune response and results in thedevelopment of a solid caseous tubercle surrounding the area of infectedcells. Tuberculosis bacilli possess many potential T-cell antigens andseveral have now been identified [Andersen 1994, Dan. Med. Bull. 41,205]. Some of these antigens are secreted by the bacteria. Continued DTHliquefies the tubercle, thereby releasing entrapped tuberculosisbacilli. The large dose of extracellular tuberculosis bacilli triggersfurther DTH, causing damage to the bronchi and dissemination bylymphatic, hematogenous and bronchial routes, and eventually allowinginfectious bacilli to be spread by respiration.

Cell-mediated immunity to tuberculosis involves several types of immuneeffector cells. Activation of macrophages by cytokines, such asinterferon-.gamma., represents an effective means of minimizingmacrophage-based intracellular mycobacterial multiplication. However,this does not lead to complete eradication of the bacilli. Acquisitionof protection against tuberculosis additionally requires T lymphocytes.Among these, T cells of both the CD8+ and CD4+ lineage appear to beparticularly important [Orme et al, 1993, J. Infect. Dis. 167, 1481].These T-cells secrete interferon-.gamma. in response to mycobacteria,indicative of a T₁ immune response, and possess cytotoxic activity tomycobacteria-pulsed target cells. In recent studies using .beta.-2microglobulin- and CD8-deficient mice, cytotoxic T lymphocyte (CTL)responses have been shown to be critical in providing protection againstM. tuberculosis [Flynn et al, 1992, Proc. Natl. Acad. Sci. USA 89,12013; Flynn et al, 1993, J. Exp. Med. 178, 2249; Cooper et al, 1993, J.Exp. Med. 178, 2243]. In contrast, B lymphocytes do not appear to beinvolved, and passive transfer of anti-mycobacterial antibodies does notprovide any protective immunity. Thus, an effective vaccine regimenagainst tuberculosis must trigger cell-mediated immune responses.

Although commonly thought of only as a pulmonary infection, TB is wellknown to afflict many parts of the body. In addition to pulmonary TB,examples of other foci of tubercular infection include miliary TB(generalized hematogenous or lymphohematogenous TB), central nervoussystem TB, pleural TB, TB pericarditis, genitourinary TB, TB of thegastrointestinal tract, TB peritonitis, TB of the adrenals, TB of theliver, TB of the bones and joints (for example, TB spondylitis or Pott'sDisease), TB lymphadenitis, and TB of the mouth, middle ear, larynx, andbronchial tree.

Conventional therapy for TB includes treatment with regimens containingpyrazinamide, isoniazid, ethambutol, streptomycin, rifampin, rifabutin,clarithromycin, ciprofloxacin, clofazamine, azithromycin, ethionamide,amikacin and resorcinomycin A. To treat latent (inactive) TB infection,isoniazid may be used alone. However, the usual initial treatment forpulmonary tuberculosis includes isoniazid in combination with at leastone other drug, such as ethambutol, streptomycin, rifampin orethionamide. Retreatment of pulmonary tuberculosis typically involvesdrug combinations including rifampin and other drugs as noted above.Development of resistance of the causative agent to anti-TB drugs,especially isoniazid, is well known. Extrapulmonary tuberculosis is alsousually treated with a combination including rifampin and at least oneof the other three drugs mentioned.

Mycobacterium avium Complex (MAC)

M. avium and M. intracellulare are members of the Mycobacterium aviumcomplex (MAC). M. paratuberculosis is a subspecies of M. avium and isalso generally included in the MAC. These species have becomeincreasingly important in recent years because of the high prevalence ofdisseminated MAC infection in AIDS patients. The Mycobacterium aviumcomplex is comprised of 28 serovars which are distinguishable on thebasis of their biochemical and seroagglutination characteristics (seereview by Inderlied, et al. 1993. Clin. Microbial. Rev. 6, 266-310).Depending on the method of classification, 10-12 of the 28 serovars areclassified as belonging to the species Mycobacterium avium, and 10-12belong to the species Mycobacterium intracellulare. Six of the MACserovars have not yet been definitively classified. MAC infectionscurrently account for approximately 50% of the pathogenic isolatesidentified by mycobacteriology labs and are most common among AIDS andother immuno-compromised patients. Early diagnosis and treatment of MACinfections can improve and prolong the lives of infected individuals.

Anthrax and Anthrax Toxin

Anthrax toxin, produced by the gram positive rod-shaped aerobic,spore-forming bacterium Bacillus anthracis, is the toxic virulencefactor secreted by this organism. B. anthracis is often considered foruse as a biological weapon due to the potency of the secreted exotoxin,and to the capacity of the bacterium to form dormant spores which resistharsh environmental conditions. Sporulation enables ready transport anddistribution of large quantities of toxin-producing bacteria. The toxinis actually a composite consisting of 3 separate secreted proteins fromthe bacterium. The 3 proteins are protective antigen (PA), lethal factor(LF), and edema factor (EF). While LF and EF directly damage cells andcause disease, the PA is the focus of this disclosure. PA is crucial tothe virulence of anthrax toxin, since the PA molecule is designed toimport both LF and EF inside the membranes of cells. In the absence ofPA-induced intracellular transport, anthrax toxin is unable to effecttissue destruction, since LF and EF only function from within the cell.The importance of PA in the function of anthrax toxin is underscored bythe effective use of PA as the immunogen in anthrax vaccine. Bygenerating an immune response against PA, the vaccine confers protectionagainst full (3 component) anthrax toxin.

A closer examination of the interaction between PA and the host cellsattacked by anthrax toxin is instructive. PA is first secreted as an 83kDa monomeric polypeptide by B. anthracis in a large and functionallyinactive form. This inactive PA binds to a mammalian receptor on thesurface of host cells. The PA receptor has recently been isolated andsequenced, and found to possess von Willebrand Factor-like regions.After docking on the surface of host cells, PA interacts with a proteasepresent on the cell surface. The protease processes the large andinactive PA molecule into a smaller and active 63 kDa fragment. TheC-terminal 63 kDa fragment (PA63) remains bound to the cell and theN-terminal 20 kDa (PA20) dissociates from PA63. The identity of theprotease has been the focus of scant research effort, and it is poorlycharacterized. However, prior studies have shown that the protease hascharacteristics that suggest it is a host-derived serine protease. Apossible serine protease candidate noted in the literature is related tofurine (itself a serine protease), but other serine proteases, such aselastase, proteinase-3, clostripain, or trypsin are possiblealternatives (Molloy, S. S. et al. J Biol Chem 267, 16396-16402 (1992)).This proteolytic cleavage and subsequent dissociation of PA20 confer twonew properties on PA63: (1) the ability to oligomerize into aring-shaped heptameric SDS-dissociable structure termed prepore and (2)the ability to bind EF and LF. Oligomers containing PA63-EF, PA63-LF, ora combination of PA63-EF and PA63-LF are endocytosed and trafficked toan acidic compartment, where the PA63 prepore inserts into the membraneand forms a pore. During or after pore formation, EF and LF aretranslocated across the endosomal membrane into the cytoplasm. EF is acalmodulin-dependent adenylate cyclase which may protect the bacteriafrom destruction by phagocytes. LF is a metalloprotease that can killmacrophages or, at lower concentrations, induce macrophages tooverproduce cytokines, possibly resulting in death of the host. Theseheptamers function as the transport vehicle to deliver LF and EF insideof the cell. Once inside the cell, LF and EF initiate abnormalities incell function.

Because of some of the difficulties and inadequacies of conventionaltherapy for tuberculosis, other mycobacterial infections, and anthrax,new therapeutic modalities are desirable.

The inventor discloses a novel method of use for serine proteaseinhibitors as therapeutic agents to treat infections caused bytuberculosis (TB) and Mycobacterium avium complex (MAC). These areintracellular human pathogens that establish infection and prolongedlatency by infecting and surviving within human macrophages. Therefore,blocking the internalization of TB or MAC within macrophages is a novelapproach to therapy vs these infectious agents. In an infectivity assay,the inventors have shown that α1-antitrypsin significantly inhibitedboth TB and MAC infection of human monocyte-derived-macrophages (MDM).

A novel approach to nullify the action of anthrax toxin is to blockaccess of the toxin to the interior of the cell by interfering with theaction of the host-derived serine protease that resides on the cellsurface.

This invention thus addresses a long-felt need for safe and effectivemethods of treatment of tuberculosis, other mycobacterial infections,other Gram negative and Gram positive bacterial infections, and anthrax.

SUMMARY OF THE INVENTION

The present invention provides methods for treating bacterial infectionsin a mammal comprising administering to a subject in need thereof of atherapeutically effective amount of a composition comprising a substanceexhibiting mammalian α1-antitrypsin or inhibitor of serine proteaseactivity or a functional derivative thereof, and a pharmaceuticallyacceptable excipient.

In one embodiment, the bacterial infections that may be treated orameliorated using the compositions and methods of the invention arethose infections caused by Gram negative bacterial organisms comprisingN. gonorrhoeae, N. meningitidis, M. catarrhalis, H. influenzae, E. coli,all Klebsiela spp., all Enterobacter spp., all Serratia spp., allSalmonella spp., all Shigella spp., Proteus mirabilis, Proteus vulgaris,all Providencia spp., all Morganella spp., all Citrobacter spp., allAeromonas spp., all Acinetobacter spp., Pseudomonas aeruginosa, allPasteurella spp., Pseudomonas cepacia, Stenotrophomonas maltophilia, Y.enterocolitica and other Yersinoiiosis, all Legionella spp., P.multocida, H. ducreyeii, all Chlamyidia spp., Mycoplasma pneumoniae,Mycoplasma hominis, Bacteroides fragilis, P. melaninogenica, allMoraxella spp., all Bortedella spp., or any combination thereof.

In another embodiment, the bacterial infections that may be treated orameliorated using the compositions and methods of the invention arethose infections caused by Gram positive bacterial organisms comprisingC. tetani, C. botulinum, C. difficile, Group A, B C, and GStreptococcus, Streptococcus pneumoniae, Streptococcus milleri group,Viridans streptococcus, all Listeria spp., all Staphylococcus spp., S.aureus (MSSA), S. aureus (MRSA), S. epidermidis, Enterococcus faecalis,Enterococcus faecium, all Clostridium spp. including C. diptheriea, C.jeikium, all Rhodococcus spp., all Leukonostoc spp., or any combinationthereof.

In yet another embodiment, the bacterial infections that may be treatedor ameliorated using the compositions and methods of the invention arethose infections caused by acid fast bacilli comprising Mycobacteriumtuberculosis, and atypical Mycobacteria (M. avium, M. intracelllulare,M. kansasii, M. chelonei, M. fortuitum, M. scrofulaceum, M. ulceranis,M. leprae, M. xenopi, M. bovis, M. gordonae, M. haemophilum, M. marinum,M. genavense, M. avium and intracellulari, and M. simiae), or anycombination thereof.

The present invention provides methods for treating mycobacterialinfections in a mammal comprising administering to a subject in needthereof a therapeutically effective amount of a composition comprising asubstance exhibiting mammalian α1-antitrypsin or inhibitor of serineprotease activity or a functional derivative thereof; and apharmaceutically acceptable excipient.

In one embodiment, the Mycobacterium inhibited from infectingmacrophages comprises a Mycobacterium from the genus Mycobacterium thatincludes M. tuberculosis M. bovis, M. leprae, M. avium-intracellulare,M. chelonei (also known as borstelense and abscessus), M. africanum, M.marinium (also known as balnei and platypoecilus), M. buruli (also knownas ulcerans), M. fortuitum (also known as giae, minetti, and ranae), M.haemophilum, M. intracellulare, M. kansasii (also known as luciflavum),M. littorale (also known as xenopi), M. malmoense, M. marianum (alsoknown as scrofulaceum and paraffinicum), M. simiae, M. szulgai, M.ulcerans, M. avium (also known as brunense), M. flavascens, M.lepraemurium, M. microti, M. paratuberculosis (which is the causativeagent for Johne's Disease), M. gordonae (also known as aquae), M.gastri, M. phlei (also known as moelleri and as timothy bacillus), M.nonchromogenicum, M. smegmatis, M. terrae, M. triviale, and M. vaccae,or any combination thereof.

In another embodiment, the Mycobacterium inhibited from infectingmacrophages comprises a Mycobacterium from the genus Mycobacterium thatincludes non-tuberculosis mycobacteria that are divided into four groupscomprising Runyon groups, selected from the group consisting of Group I(slow-growing photochromogens), Group II (slow-growing scotochromogens),Group III (slow-growing nonphotochromogens), and Group IV(rapidly-growing mycobacteria), or any combination thereof.

Therefore, in one aspect, the present invention provides methods oftreating mycobacterial diseases dependent on the infection ofmacrophages.

Also provided is a method of inhibiting mycobacterial infection ofmacrophages, which comprises administering to a mammal susceptible tomycobacterial infection of macrophages an effective amount of asubstance exhibiting mammalian α1-antitrypsin or inhibitor of serineprotease activity. Without limiting to α1-antitrypsin, the substance maybe a compound that inhibits proteinase-3, cathepsin G, elastase, or anyother serine protease.

In a preferred embodiment the agent that inhibits mycobacterialinfection of human monocyte-derived-macrophages comprisesα1-antitrypsin. In addition, peptides of interest are homologous andanalogous peptides. While homologues are natural peptides with sequencehomology, analogues will be peptidyl derivatives, e.g., aldehyde orketone derivatives of such peptides. Typical examples of analogues areTLCK or TPCK. Without limiting to α1-antitrypsin and peptide derivativesof α1-antitrypsin, compounds like oxadiazole, thiadiazole, CE-2072,UT-77, and triazole peptoids are preferred.

The agent that inhibits mycobacterial infection of humanmonocyte-derived-macrophages can also be an inhibitor of serine proteaseactivity, an inhibitor of elastase, or an inhibitor of proteinase-3. Theinhibitor of serine protease activity can include, but is not limitedto, small organic molecules including naturally-occurring, synthetic,and biosynthetic molecules, small inorganic molecules includingnaturally-occurring and synthetic molecules, natural products includingthose produced by plants and fungi, peptides, variants ofα1-antitrypsin, chemically modified peptides, and proteins. An inhibitorof serine protease activity has the capability of inhibiting theproteolytic activity of trypsin, elastase, kallikrein, and/or otherserine proteases.

Also contemplated within the scope of the present invention is a methodof preventing a deficiency of functional endogenous α1-antitrypsinlevels in a patient susceptible to a mycobacterial infection ofmacrophages that is mediated by endogenous host serine protease orserine protease-like activity, by treating with a pharmaceuticalcomposition in a pharmaceutically acceptable carrier comprisingeffective amounts of a substance exhibiting mammalian α1-antitrypsin orinhibitor of serine protease activity. The pharmaceutical compositioncan be a peptide or a small molecule, which exhibits α1-antitrypsin orinhibitor of serine protease activity.

In yet another aspect, the present invention provides a method forpreventing a symptom of anthrax in a subject thought to be at risk forexposure to Bacillus anthracis comprising administering to the subject apharmaceutically effective amount of a substance exhibiting mammalian“1-antitrypsin or inhibitor of serine protease activity, wherein saidmammalian “1-antitrypsin or inhibitor of serine protease activitysubstance inhibits the endogenous host protease cell-surface processingof inactive large PA into the active smaller PA molecule, and wherein ifthe subject is exposed to Bacillus anthracis, a symptom of said exposureis prevented.

In another aspect, the present invention provides a method forpreventing a symptom of anthrax in a subject suspected of having beenexposed to Bacillus anthracis comprising administering to the subject apharmaceutically effective amount of a substance exhibiting mammalianα1-antitrypsin or inhibitor of serine protease activity, wherein saidmammalian α1-antitrypsin or inhibitor of serine protease activitysubstance inhibits the endogenous host protease cell-surface processingof inactive large PA into the active smaller PA molecule, and wherein ifthe subject is exposed to Bacillus anthracis, a symptom of said exposureis prevented.

In another aspect, the present invention provides a method forameliorating a symptom of anthrax in a subject in need of saidamelioration comprising administering to the subject a pharmaceuticallyeffective amount of a substance exhibiting mammalian α1-antitrypsin orinhibitor of serine protease activity, wherein said mammalianα1-antitrypsin or inhibitor of serine protease activity substanceinhibits the endogenous host protease cell-surface processing ofinactive large PA into the active smaller PA molecule.

In the above-recited methods, the symptom of anthrax that is inhibitedor prevented is selected from the group consisting of cutaneousulceration, edema, and scar formation, or any combination thereof.

In one embodiment, the methods of the present invention are used toprevent or ameliorate a symptom of cutaneous, gastrointestinal, and/orinhalation anthrax. In one embodiment, the methods of the presentinvention are used to prevent or ameliorate a symptom of anthraxselected from the group consisting of malaise, fever, dry cough,myalgias, and chest pains, ventilatory compromise, sweating, widening ofthe mediastimum on radiographic studies, edema of the neck and chest,necrotizing mediastinal lymphadenitis, non-pitting edema, eschar,nausea, vomiting, fever, abdominal pain, bloody diarrhea, mucosalulcerations, hemorrhagic mesenteric lymphadenitis, or any combinationthereof.

In yet another aspect, the present invention is directed to a method ofrelieving or ameliorating the pain or symptoms associated with any oneor more of the above-identified bacterial diseases or indications,mycobacterial diseases or indications, or anthrax infection in a mammalsuffering from any one or more of the above-identified bacterialdiseases or indications, mycobacterial diseases or indications, oranthrax infection which comprises administering to the mammal in needthereof a therapeutically effective pain or symptom-reducing amount of apharmaceutical composition comprising effective amounts of a substanceexhibiting mammalian α1-antitrypsin or inhibitor of serine proteaseactivity, either alone or in combination with one or moreanti-inflammatory compounds or immunomodulatory agents; and apharmaceutically acceptable carrier or excipient, wherein said mammalianα1-anitrypsin or inhibitor of serine protease activity substance issufficient to inhibit or ameliorate the bacterial disease or indication,mycobacterial disease or indication, or anthrax infection of the host.

In one embodiment, the reduction or inhibition of pain and/or symptomsassociated with one or more of each of the above-recited mycobacterialindications, bacterial infections or anthrax infections is on the orderof about 10-20% reduction or inhibition. In another embodiment, thereduction or inhibition of pain is on the order of 30-40%. In anotherembodiment, the reduction or inhibition of pain is on the order of50-60%. In yet another embodiment, the reduction or inhibition of thepain associated with each of the recited indications is on the order of75-100%. It is intended herein that the ranges recited also include allthose specific percentage amounts between the recited range. Forexample, the range of about 75 to 100% also encompasses 76 to 99%, 77 to98%, etc., without actually reciting each specific range therewith.

Accordingly, the overall aspect of the present invention to providecompounds that exhibit inhibitory activity toward serine proteases.Thus, it should be recognized that this invention is applicable to thecontrol of catalytic activity of serine proteases in any appropriatesituation including, but not necessarily limited to, medicine, biology,agriculture, and microbial fermentation.

One aspect of the present invention is to provide clinically acceptableserine protease inhibitors with recognized utility and exhibitingrelatively high activity at relatively low concentrations.

In one embodiment, the α1-antitrypsin used in the methods andcompositions of the present invention comprises Aralast® (Baxter;alpha₁-proteinase inhibitor (human)), Zemaira® (Aventis Behring;alpha₁-proteinase inhibitor (human)), and Prolastin® (Bayer;alpha₁-proteinase inhibitor (human)), or any combination thereof.

The present invention provides methods for therapeutically orprophylactically treating bacterial infections in a subject.

The method for therapeutically treating bacterial or mycobacterialinfections comprises the step of administering pharmaceuticallyeffective amounts of a substance exhibiting mammalian α1-antitrypsin orinhibitor of serine protease activity or derivative thereof to thesubject after occurrence of the bacterial or mycobacterial disease.

The method for prophylactically treating bacterial or mycobacterialinfections comprises the step of administering pharmaceuticallyeffective amounts of a substance exhibiting mammalian α1-antitrypsin orinhibitor of serine protease activity or derivative thereof to thesubject prior to the occurrence of the bacterial or mycobacterialdisease.

Either methodology inhibits the bacterial infection or the mycobacterialinfection of macrophages.

For each of the above-recited methods of the present invention, thetherapeutically effective amount of one or more substances exhibitingmammalian α1-antitrypsin or inhibitor of serine protease activity or afunctional derivative thereof may be administered to a subject in needthereof in conjunction with a therapeutically effective amount of one ormore anti-microbacterial drugs and/or inflammatory compounds and/or atherapeutically effective amount of one or more immunomodulatory agents.

In certain embodiments of the method of the present invention, theanti-inflammatory compound or immunomodulatory drug comprisesinterferon; interferon derivatives comprising betaseron, β-interferon;prostane derivatives comprising iloprost, cicaprost; glucocorticoidscomprising cortisol, prednisolone, methylprednisolone, dexamethasone;immunsuppressives comprising cyclosporine A, FK-506, methoxsalene,thalidomide, sulfasalazine, azathioprine, methotrexate; lipoxygenaseinhibitors comprising zileutone, MK-886, WY-50295, SC-45662, SC-41661A,BI-L-357; leukotriene antagonists; peptide derivatives comprising ACTHand analogs thereof; soluble TNF-receptors; TNF-antibodies; solublereceptors of interleukines, other cytokines, T-cell-proteins; antibodiesagainst receptors of interleukines, other cytokines, T-cell-proteins;and calcipotriols and analogues thereof taken either alone or incombination.

The present invention also relates to the combined use of thepharmaceutical composition exhibiting mammalian α1-antitrypsin orinhibitor of serine protease activity in combination with one or moreantibacterial or antiviral compositions or any combination thereof fortreating any one of the aforementioned bacterial or mycobacterialdiseases, or any combination thereof.

In each of the above-recited methods, the mammalian α1-antitrypsin orinhibitor of serine protease activity substance may be part of a fusionpolypeptide, wherein said fusion polypeptide comprises mammalianα1-antitrypsin or a substance with inhibitor of serine protease activityand an amino acid sequence heterologous to said mammalian α1-antitrypsinor inhibitor of serine protease activity substance.

In certain embodiments, the fusion polypeptide contemplated for use inthe methods of the present invention comprise a human immunoglobulinconstant region, such as for example, a human IgG1 constant region,including a modified human IgG1 constant region wherein the IgG1constant region does not bind the Fc receptor and/or does not initiateantibody-dependent cellular cytotoxicity (ADCC) reactions.

In yet other embodiments, the fusion polypeptide contemplated for use inthe methods of the present invention can additionally comprise an aminoacid sequence that is useful for identifying, tracking or purifying thefusion polypeptide, e.g., the fusion polypeptide can further comprise aFLAG or HIS tag sequence. The fusion polypeptide can additionallyfurther comprise a proteolytic cleavage site which can be used to removethe heterologous amino acid sequence from the mammalian α1-antitrypsinor the substance with inhibitor of serine protease activity. In each ofthe above-recited compositions and methods of the invention the agentthat inhibits the bacterial infection, mycobacterial infection of humanmonocyte-derived-macrophages or anthrax comprises α1-antitrypsin. Inaddition, peptides of interest are homologous and analogous peptides.While homologues are natural peptides with sequence homology, analogueswill be peptidyl derivatives, e.g., aldehyde or ketone derivatives ofsuch peptides. Typical examples of analogues are TLCK or TPCK. Withoutlimiting to α1-antitrypsin and peptide derivatives of α1-antitrypsin,compounds like oxadiazole, thiadiazole, CE-2072, UT-77, and triazolepeptoids are preferred.

In other embodiments, the agent that inhibits the bacterial infection,the mycobacterial infection of human monocyte-derived-macrophages and/oranthrax can also be an inhibitor of serine protease activity, aninhibitor of elastase, or an inhibitor of proteinase-3. The inhibitor ofserine protease activity can include, but is not limited to, smallorganic molecules including naturally-occurring, synthetic, andbiosynthetic molecules, small inorganic molecules includingnaturally-occurring and synthetic molecules, natural products includingthose produced by plants and fungi, peptides, variants ofα1-antitrypsin, chemically modified peptides, and proteins. An inhibitorof serine protease activity has the capability of inhibiting theproteolytic activity of trypsin, elastase, kallikrein, and/or otherserine proteases.

In one embodiment of the invention, the peptide can be protected orderivatized in various ways, e.g., N-terminal acylation, C-terminalamidation, cyclization, etc. In a specific embodiment, the N-terminus ofthe peptide is acetylated.

The peptides of interest are homologous and analogous peptides. Whilehomologues are natural peptides with sequence homology, analogues willbe peptidyl derivatives, e.g., aldehyde or ketone derivatives of suchpeptides. Without limiting to AAT and peptide derivatives of AAT, thecompounds like oxadiazole, thiadiazole and triazole peptoids andsubstances comprising certain phenylenedialkanoate esters are preferred.

In each of the above-recited methods, the mammalian α1-antitrypsin orinhibitor of serine protease activity substance contemplated for usewithin the methods of the present invention further comprises a seriesof peptides comprising carboxyterminal amino acid peptides correspondingto AAT. These pentapeptides can be represented by a general formula (I):I-A-B-C-D-E-F-G-H-II, wherein I is Cys or absent; A is Ala, Gly; Val orabsent; B is Ala, Gly, Val, Ser or absent; C is Ser, Thr or absent; D isSer, Thr, Ans, Glu, Arg, Ile, Leu or absent; E is Ser, Thr, Asp orabsent; F is Thr, Ser, Asn, Gln, Lys, Trp or absent; G is Tyr or absent;H is Thr, Gly, Met, Met(O), Cys, Thr or Gly; and II is Cys, an amidegroup, substituted amide group, an ester group or absent, wherein thepeptides comprise at least 4 amino acids and physiologically acceptablesalts thereof. Among this series of peptides, several are equallyacceptable including FVFLM (SEQUENCE ID NO. 1), FVFAM (SEQUENCE ID NO.2), FVALM (SEQUENCE ID NO. 3), FVFLA (SEQUENCE ID NO. 4), FLVFI(SEQUENCE ID NO. 5), FLMII (SEQUENCE ID NO. 6), FLFVL (SEQUENCE ID NO.7), FLFVV (SEQUENCE ID NO. 8), FLFLI (SEQUENCE ID NO. 9), FLFFI(SEQUENCE ID NO. 10), FLMFI (SEQUENCE ID NO. 11), FMLLI (SEQUENCE ID NO.12), FIIMI (SEQUENCE ID NO. 13), FLFCI (SEQUENCE ID NO. 14), FLFAV(SEQUENCE ID) NO. 15), FVYLI (SEQUENCE ID NO. 16), FAFLM (SEQUENCE IDNO. 17), AVFLM (SEQUENCE ID NO. 18), and any combination thereof.

In yet another embodiment, these peptides can be represented by ageneral formula (II): NT-X1-X2-X3-X4-X5-CT or a physiologicallyacceptable salt thereof, in which NT comprises an amino acid residuepositioned at the peptide's N-terminal end, including C, an acetylgroup, or a succinyl group, provided that NT can also be absent; X1comprises an amino acid residue, including F or A; X2 comprises an aminoacid residue, including C, V, L, M, I, A, C, or S; X3 comprises an aminoacid residue, including F, A, V, M, L, I, Y, or C; X4 comprises an aminoacid residue, including L, A, F, I, V, M, C, G, or S; X5 comprises anamino acid residue, including M, A, I, L, V, F, or G; and CT comprisesan amino acid residue positioned at the peptide's C-terminal end,including C, an amide group, a substituted amide group, or an estergroup, provided that CT can also be absent, and in which the amino acidresidue can be either an L- or a D-stereoisomeric configuration. Thesepeptides comprise at least 5 amino acids and physiologically acceptablesalts thereof. Amino acids in the formula are abbreviated as 1-letterand corresponding 3-letter codes are as follow: Alanine is A or Ala;Arginine R or Arg, Asparagine N or Asn; Aspartic acid D or Asp; CysteineC or Cys; Glutamine Q or Gln; Glutamic acid E or Glu; Glycine G or Gly;Histidine H or His; Isoleucine I or Ile; Leucine L or Leu; Lysine K orLys; Methionine M or Met; Phenylalanine F or Phe; Proline P or Pro;Serine S or Ser; Threonine T or Thr; Tryptophan W or Tip; Tyrosine Y orTyr; and Valine V or Val.

In each of the above-recited methods, the mammalian α1-antitrypsin orinhibitor of serine protease activity substance contemplated for usewithin the methods of the present invention further comprises a seriesof peptides comprising amino acid peptides corresponding to portions orfragments of AAT. For example, and not by way of limitation, amino acidpeptides corresponding to 10 amino acid fragments of AAT arespecifically contemplated for use in the composition and methods of thepresent invention. In particular, amino acid peptides include MPSSVSWGIL(SEQUENCE ID NO. 19); LAGLCCLVPV (SEQUENCE II) NO. 20) SLAEDPQGDA(SEQUENCE ID NO. 21); AQKTDTSHHD (SEQUENCE ID NO. 22) QDHPTFNKIT(SEQUENCE ID NO. 23); PNLAEFAFSL (SEQUENCE ID NO. 24); YRQLAHQSNS(SEQUENCE ID NO. 25); TNIFFSPVSI (SEQUENCE ID NO. 26); ATAFAMLSLG(SEQUENCE ID NO. 27); TKADTHDEIL (SEQUENCE ID NO. 28); EGLNFNLTEI(SEQUENCE ID NO. 29); PEAQIHEGFQ (SEQUENCE ID) NO. 30); ELLRTLNQPD(SEQUENCE ID NO. 31); SQLQLTTGNG (SEQUENCE ID NO. 32); LFLSEGLKLV(SEQUENCE ID NO. 33); DKFLEDVKKL (SEQUENCE ID NO. 34); YHSEAFTVNF(SEQUENCE ID NO. 35); GDHEEAKKQI (SEQUENCE ID NO. 36); NDYVEKGTQG(SEQUENCE ID NO. 37); KIVDLVKELD (SEQUENCE ID NO. 38); RDTVFALVNY(SEQUENCE ID NO. 39); IFFKGKWERP (SEQUENCE ID NO. 40); FEVKDTEDED(SEQUENCE ID NO. 41); FHVDQVTTVK (SEQUENCE ID NO. 42); VPMMKRLGMF(SEQUENCE ID NO. 43); NIQHCKKLSS (SEQUENCE ID NO. 44); WVLLMKYLGN(SEQUENCE ID NO. 45); ATAIFFLPDE (SEQUENCE ID NO. 46); GKLQHLENEL(SEQUENCE ID NO. 47); THDIITKFLE (SEQUENCE ED NO. 48); NEDRRSASLH(SEQUENCE ID NO. 49); LPKLSITGTY (SEQUENCE ID NO. 50); DLKSVLGQLG(SEQUENCE ID NO. 51); ITKVFSNGAD (SEQUENCE ID NO. 52); LSGVTEEAPL(SEQUENCE ID NO. 53); KLSKAVHKAV (SEQUENCE ID NO. 54); LTIDEKGTEA(SEQUENCE ID NO. 55); AGAMFLEAIP (SEQUENCE ID NO. 56); MSIPPEVKFN(SEQUENCE ID NO. 57); KPFVFLMIEQ (SEQUENCE ID NO. 58); NTKSPLFMGK(SEQUENCE ID NO. 59); VVNPTQK (SEQUENCE ID NO. 60), or any combinationthereof. It is specifically intended that the AAT peptides recitedcontemplated for use in the compositions and methods of the presentinvention are also intended to include any and all of those specific AATpeptides other than the 10 amino acid AAT peptides of SEQ ID NO. 63depicted supra. For example, while AAT peptides including amino acids1-10, amino acids 11-20, amino acids 21-30, etc. of SEQ ID NO. 63 havebeen enumerated herein, it is intended that the scope of thecompositions and methods of use thereof specifically include all of thepossible combinations of AAT peptides such as amino acids 2-12, aminoacid 3-13, 4-14, etc. of SEQ ID NO. 63, as well as any and all AATpeptide fragments corresponding to select amino acids of SEQ ID NO. 61,without actually reciting each specific AAT peptide of SEQ ID NO. 61therewith. Thus, by way of illustration, and not by way of limitation,Applicants are herein entitled to possession of compositions based uponany and all AAT peptide variants based upon the amino acid sequencedepicted in SEQ ID NO. 61 and use of such compositions in the methods ofthe present invention.

The AAT and similarly active compounds contemplated for use in thecompositions and methods of the present invention may be identified by aseries of assays wherein a compound (AAT) will exhibit inhibitoryactivity versus control in an assay. One of these assays comprisesblocking infection of human monocyte derived macrophages in an in vitromodel of infection as described in detail in Example 1 of the detaileddescription of this disclosure.

In one embodiment, with respect to the use of the compositions andmethods of the present invention to prevent or ameliorate a symptomcaused by either Bacillus anthracis, Corynebacterium diptheriae, orPseudomonas aeruginosa, specifically excluded within the scope of thepresent invention are those furin endoprotease inhibitors comprising anα1-antitrypsin variant having an amino acid sequence comprising theamino acids of the native α1-antitrypsin molecule, except that thesequence at position 355-358 of the native protein (-Ala-Ile-Pro-Met-)is changed to the novel sequence -Arg-X-X-Arg-, wherein X is any aminoacid, at positions 355-358 of the native α1-antitrypsin amino acidsequence as disclosed in U.S. Pat. Nos. 5,604,201 and 6,022,855.

Also specifically excluded within the scope of the compositions andmethods of the present invention to prevent or ameliorate a symptomcaused by either Bacillus anthracis, Corynebacterium diptheriae, orPseudomonas aeruginosa are those α1-antitrypsin Portland variantswherein the amino acid sequence at positions 355-358 of theα1-antitrypsin amino acid Portland sequence is -Arg-Ile-Pro-Arg- asdisclosed in U.S. Pat. Nos. 5,604,201 and 6,022,855.

Also specifically excluded within the scope of the compositions andmethods of the present invention to prevent or ameliorate a symptomcaused by either Bacillus anthracis, Corynebacterium diptheriae, orPseudomonas aeruginosa are peptides having amino acid sequences of about4 to about 100 amino acids in length comprising the amino acid sequence-Arg-Xaa-Xaa-Arg-, wherein each Xaa is any amino acid as is disclosed inU.S. Pat. Nos. 5,604,201 and 6,022,855.

In yet another embodiment, with respect to the use of the compositionsand methods of the present invention to prevent or ameliorate a symptomof anthrax, specifically excluded within the scope of the presentinvention are those furin endoprotease inhibitors comprising HexArg asdisclosed in Miroslav S. Sarac et al. (Infection and Immunity, January2004, p. 602-605, Vol. 72, No. 1 Protection against Anthrax Toxemia byHexa-D-Arginine In Vitro and In Vivo).

The invention further provides pharmaceutical compositions comprisingsuch agents.

The preferred doses for administration can be anywhere in a rangebetween about 10 ng and about 10 mg per ml or mg of the formulation. Thetherapeutically effective amount of AAT peptides or drugs that havesimilar activities as AAT or peptides drug can be also measured in molarconcentrations and may range between about 1 nM and about 10 mM. Theformulation is also contemplated in combination with a pharmaceuticallyor cosmetically acceptable carrier. The precise doses can be establishedby well-known routine clinical trials without undue experimentation.

In one aspect of the invention, the pharmaceutical compositions of thepresent invention are administered orally, systemically, via an implant,intravenously, topically, intrathecally, intracranially,intraventricularly, by inhalation or nasally.

In certain embodiments of the methods of the present invention, thesubject or mammal is a human.

In other embodiments of the methods of the present invention, thesubject or mammal is a veterinary and/or a domesticated mammal.

There has been thus outlined, rather broadly, the important features ofthe invention in order that a detailed description thereof that followscan be better understood, and in order that the present contribution canbe better appreciated. There are additional features of the inventionthat will be described hereinafter.

In this respect, before explaining at least one embodiment of theinvention in detail, it is to be understood that the invention is notlimited in its application to the details as set forth in the followingdescription and figures. The present invention is capable of otherembodiments and of being practiced and carried out in various ways.Additionally, it is to be understood that the terminology andphraseology employed herein are for the purpose of description andshould not be regarded as limiting.

As such, those skilled in the art will appreciate that the conception,upon which this disclosure is based, can readily be used as a basis fordesigning other methods for carrying out the several features andadvantages of the present invention. It is important, therefore, thatthe claims be regarded as including such equivalent constructionsinsofar as they do not depart from the spirit and scope of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effect of α-1-antitrypsin (AAT) and AAT mimic onMycobacterium avium complex (mac) infection of human monocyte-derivedmacrophages (n=4).

FIG. 2 illustrates the effect of α-1-antitrypsin (AAT) and AAT mimic onMycobacterium avium complex (mac)-induced TNFα in human monocyte-derivedmacrophages.

FIG. 3 illustrates the effect of α-1-antitrypsin (AAT) and AAT mimic onMycobacterium avium complex (mac)-induced TNFα in human monocyte-derivedmacrophages: time-course experiment (n=1).

FIGS. 4A-4H illustrate the Bacillus anthracis toxin mechanism and themethod by which serine protease inhibitors neutralize the toxin.

FIG. 5 illustrates the effect of α-1-antitrypsin on stimulatedinterleukin-1 beta production in whole human blood.

DETAILED DESCRIPTION OF THE INVENTION

Standard Methods

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition 1989, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Animal Cell Culture,R. I. Freshney, ed., 1986).

Therapeutic Methods

The present invention provides methods for treating mycobacterialinfections comprising administering to a subject in need thereof of atherapeutically effective amount of a composition comprising aneffective amount of a substance exhibiting mammalian α1-antitrypsin orinhibitor of serine protease activity or a functional derivativethereof; and a pharmaceutically acceptable excipient.

According to the methods of the present invention, mycobacterialinfection of macrophages is inhibited to obtain important therapeuticbenefits.

Therefore, administration of a dosage of the invention composition,i.e., α1-antitrypsin, or a fragment, derivative or analog thereof, canbe beneficial for the treatment of mycobacterial diseases or disorders.In a preferred aspect, the agent is an analog of α1-antitrypsin that cancross the blood brain barrier, which would allow for intravenous or oraladministration. Many strategies are available for crossing the bloodbrain barrier, including but not limited to, increasing the hydrophobicnature of a molecule; introducing the molecule as a conjugate to acarrier, such as transferrin, targeted to a receptor in the blood brainbarrier; and the like. In another embodiment, the agent can beadministered intracranially or, more directly, intraventricularly. Inyet another embodiment, the agent can be administered by way ofinhalation or nasally.

In a further embodiment, the methods and compositions of the inventionare useful in the therapeutic treatment of mycobacterial diseases ordisorders of the immune system. In a yet further embodiment, diseasescan be prevented by the timely administration of the agent of theinvention as a prophylactic, prior to onset of symptoms, or signs, orprior to onset of severe symptoms or signs of a mycobacterial disease.Thus, a patient at risk for a particular mycobacterial disease can betreated with serine protease inhibitors, for example,(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(3-Trifluoromethylbenzyl)-1,2,4-ox-adiazolyl)carbonyl)-2-(S)-Methylpropyl]-L-Prolinamide;as a precautionary measure.

The effective dose of the agent of the invention, and the appropriatetreatment regime, can vary with the indication and patient condition,and the nature of the molecule itself, e.g., its in vivo half-life andlevel of activity. These parameters are readily addressed by one ofordinary skill in the art and can be determined by routineexperimentation.

The preferred doses for administration can be anywhere in a rangebetween about 0.01 mg and about 20 mg per ml of biologic fluid oftreated patient. The therapeutically effective amount of α1-antitrypsin,peptides, or drugs that have similar activities as α1-antitrypsin orpeptides can be also measured in molar concentrations and can rangebetween about 1 nM to about 2 mM.

Serine Protease Inhibitors

It is to be understood that the present invention is not limited to theexamples described herein, and other serine proteases known in the artcan be used within the limitations of the invention. For example, oneskilled in the art can easily adopt inhibitors as described in WO98/24806, which discloses substituted oxadiazole, thiadiazole andtriazole as serine protease inhibitors. U.S. Pat. No. 5,874,585discloses substituted heterocyclic compounds useful as inhibitors ofserine proteases; including:(benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3-trifluoro-methylbenzyl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-methylpropyl]-L-prolinami-debenzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(2-phenylethyl)-1,2,4-oxadiazolyl-)carbonyl)-2-(S)-methylpropyl]-L-prolinamide;(benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(2-methoxybenzyl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-methylpropy-1]-L-prolinamide;(benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(trifluoromethyl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-methylpropyl]-L-prolinamide;(benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(methyl)-1,2,4-oxadiazolyl)carbony-1)-2-(S)-Methylpropyl]-L-Prolinamide;(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(difluoromethyl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-Methylpropyl]-L-Prol-inamide;(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(benzyl)-1,2,4-oxadiazolyl-)carbonyl)-2-(S)-Methylpropyl]-L-Prolinamide;(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(3-methoxybenzyl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-Methylpropy-1]-L-Prolinamide;(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(2,6-difluorobenz-yl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-Methylpropyl]-L-Prolinamide;(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(trans-styryl)-1,2,4-oxadiazolyl)c-arbonyl)-2-(S)-Methylpropyl]-L-Prolinamide;(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(trans-4-Trifluoromethylstyryl)-1,2,4-oxadiazolyl)carbonyl)-2-(S-)-Methylpropyl]-L-Prolinamide;(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(tra-ns-4-Methoxystyryl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-Methylpropyl]-L-Prol-inamide;(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(3-Thienylmethyl)-1,2,4-ox-adiazolyl)carbonyl)-2-(S)-Methylpropyl]-L-Prolinamide;(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(Phenyl)-1,2,4-oxadiazolyl)carbony-1)-2-(S)-methylpropyl]-L-prolinamide;and(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(3-Phenylpropyl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-Methylpropyl]-L-Prolinamide.U.S. Pat. No. 5,216,022 teaches other small molecules useful for thepractice of this invention, including:Benzyloxycarbonyl-L-valyl-N-[1-(2-[5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl-]-L-prolinamide(also known as CE-2072),Benzyloxycarbonyl-L-valyl-N-[1-(2-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolin-amide;Benzyloxycarbonyl-L-valyl-N-[-(2-(5-(methyl)-1,3,4-oxadiazoly]carbo-nyl)-2-(S)-methylpropyl]-L-prolinamide;Benzyloxycarbonyl)-L-valyl-N-[1-(2-(5-(3-trifluoromethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylprop-yl]-L-prolinamide;(Benzyloxycarbonyl)-L-valyl-N-[1-(2-(5-(4-Dimethylaminobenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;Benzyloxycarbonyl)-L-valyl-N-[1-(2-(5-(1-napthylenyl)-1,3,4-oxadiazolyl]c-arbonyl)-2-(S)-methylpropyl]-L-prolinamide;(Benzyloxycarbonyl)-L-valyl-[1-(3-(5-(3,4-methylenedioxybenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methyl-propyl]-L-prolinamide;Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3,5-dimethyl-benzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3,5-dimethoxybenzyl)-1,2,4-oxadia-zolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;(Benzyloxycarbonyl)-L-v-alyl-N-[1-(3-(5-(3,5-ditrifluoromethylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3-methylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolina-mide;(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(biphenylmethine)-1,2,4-oxadi-azolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(4-phenylbenzyl)-1,2,4-oxadiazolyl-]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3-phenylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl-]-L-prolinamide;(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3-phenoxybenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(cyclohexylmethylene)-1,2,4-oxadia-zolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;(Benzyloxycarbonyl)-L-v-alyl-N-[1-(3-(5-(3-trifluoromethyldimethylmethylene)-1,2,4-oxadiazolyl]car-bonyl)-2-(S)-methylpropyl]-L-prolinamide;(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(1-napthylmethylene)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl-]-L-prolinamide;(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3-pyridylmethyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3,5-diphenylbenzyl)-1,2,4-oxadiaz-olyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;(Benzyloxycarbonyl)-L-va-lyl-N-[1-(3-(5-(4-dimethylaminobenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-m-ethylpropyl]-L-prolinamide;2-(5-[(Benzyloxycarbonyl)amino]-6-oxo-2-(4-flu-orophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(3-(5-(3-trifluoromethylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-(S)-2-methylpropyl]acetamide;2-(5-Amino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(3-(5-(3-trifluoromethylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-acetamide;2-(5-[(Benzyloxycarbonyl)amino]-6-oxo-2-(4-fluorophenyl)-1,6-di-hydro-1-pyrimidinyl]-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbony-1)-(S)-2-methylpropyl]acetamide;2-(5-Amino-6-oxo-2-(4-fluorophenyl)-1,6-d-ihydro-1-pyrimidinyl]-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbon-yl)-2-methylpropyl]acetamide;(Pyrrole-2-carbonyl)-N-(benzyl)glycyl-N-[1-(-2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]amide-;(Pyrrole-2-carbonyl)-N-(benzyl)glycyl-N-[1-(3-(5-(3-trifluoromethylbenzy-1)]-1,2,4-oxadiazolyl)-(S)-methylpropyl]amide;(2S,5S)-5-Amino-1,2,4,5,6,7-hexahydroazepino-[3,2,1]-indole-4-one-carbonyl-N-[1-(2-(5-(3-methylbenzyl-)-1,3,4-oxadiazolyl]carbonyl)-(R,S)-2-methylpropyl]amide;BTD-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpro-pyl]amide;(R,S)-3-Amino-2-oxo-5-phenyl-1,4,-benzodiazepine-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;(Benzyloxycarbonyl)-L-valyl-2-L-(2,3-dihydro-1H-indole)-N-[1-(2-(5-(3-met-hylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]amide;(Benzyloxycarbonyl)-L-valyl-2-L-(2,3-dihydro-1H-indole)-N-[1-(3-(5-(3-tri-fluoromethylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]amide;Acetyl-2-L-(2,3-dihydro-1H-indole)-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxad-iazolyl]carbonyl)-2-(S)-methylpropyl]amide;3-(S)-(Benzyloxycarbonyl)amino-)-.epsilon.-lactam-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl-)-2-(S)-methylpropyl]acetamide;3-(S)-(Amino)-.epsilon.-lactam-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamidetrifluoroacetic acid salt; 3-(S)-[(4-morpholinocarbonyl-butanoyl)amino]-.epsilon.-lactam-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide;6-[4-Fluorophenyl]-.epsilon.-lactam-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetam-ide;2-(2-(R,S)-Phenyl-4-oxothiazolidin-3-yl]-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;2-(2-(R,S)-phenyl-4-oxothiazolidin-3-yl]-N-[1-(2-(5-(3-methylbenzyl)-1,3,-4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamide;2-(2-(R,S)-Benzyl-4-oxothiazolidin-3-yl]-N-[1-(2-(5-(3-methylbenzyl)-1,3,-4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-acetamide;2-(2-(R,S)-Benzyl-4-oxothiazolidin-3-yloxide]-N-[1-(3-(5-(3trifluorometh-ylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(R,S,)-methylpropyl]acetamide;(1-Benzoyl-3,8-quinazolinedione)-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadia-zolyl]carbonyl)-2-(S)-methylpropyl]acetamide;(1-Benzoyl-3,6-piperazinedio-ne)-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpr-opyl]acetamide;(1-Phenyl-3,6-piperazinedione)-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;[(1-Phenyl-3,6-piperazinedione)-N-[1-(3-(5-(3-trifluoromethylbenzyl)-1,2,-4-oxadiazolyl]carbonyl)]-2-(S)-methylpropyl]acetamide;3-[(Benzyloxycarbonyl)amino]-quinolin-2-one-N-[1-(2-(5-(3-methylbenzyl)-1-,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;3-[(Benzyloxycarbonyl)amino]-7-piperidinyl-quinolin-2-one-N-[1-(2-(5-(3-m-ethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;3-(Carbomethoxy-quinolin-2-one-N-[1-(2-(5-(3-methybenzyl)-1,3,4-oxadiazol-yl]carbonyl)-2-(S)-methylpropyl]acetamide;3-(Amino-quinolin-2-one)-N-[1-(-2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acet-amide;3-[(4-Morpholino)aceto]amino-quinolin-2-one-N-[1-(2-(5-(3-methylben-zyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;3,4-Dihydro-quinolin-2-one-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]-carbonyl)-2-(S)-methylpropyl]acetamide;1-Acetyl-3-(4-fluorobenzylidene)p-iperazine-2,5-dione-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl-)-2-(S)-methylpropyl]acetamide;1-Acetyl-3-(4-dimethylaminobenzylidene)piperazine-2,5-dione-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadia-zolyl]carbonyl)-2-(S)-methylpropyl]acetamide;1-Acetyl-3-(4-carbomethoxybenzylidene)piperazine-2,5-dione-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadia-zolyl]carbonyl)-2-(S)-methylpropyl]acetamide;1-Acetyl-3-[(4-pyridyl)methy-lene]piperazine-2,5-dione-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]c-arbonyl)-2-(S)-methylpropyl]acetamide;4-[1-Benzyl-3-(R)-benzyl-piperazine-2,5,-dione]-N-[1-(2-[5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-met-hylpropyl]acetamide;4-[1-Benzyl-3-(S)-benzylpiperazine-2,5,-dione]-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acet-amide;4-[1-Benzyl-3(R)-benzylpiperazine-2,5,-dione]-N-[1-(3-(5-(3-trifluo-romethylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;4-[1-Benzyl-3-(S)-benzylpiperazine-2,5,-dione]-N-[1-(3-(5-(3-trifluoromet-hylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;4-[1-Benzyl-3-(S)-benzylpiperazine-2,5,-dione]-N-[1-(3-(5-(2-dimethylami-noethyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;4-[1-Methyl-3-(R,S)-phenylpiperazine-2,5,-dione]-N-[1-(3-(5-(3-trifluorom-ethylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;4-[[-Methyl-3-(R,S)-phenylpiperazine-2,5,-dione]-N-[1-(2-(5-(3-methylben-zyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;4-[1-(4-Morpholino ethyl)3-(R)-benzylpiperazine-2,5,-dione]-N-[1-(2-(5-(-3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;5-(R,S)-Phenyl-2,4-imidazolidinedione-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-o-xadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;5-(R)-Benzyl-2,4-imidaz-olidinedione-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;5-(S)-Benzyl-2,4-imidazolidinedione-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;5-(S)-Benzyl-2,4-imidazolidinedione-N-[1-(3-(5-(3-trifluoromethylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;5-(R)-Benzyl-2,4-imidazolidinedione-N-[1-(3-(5-(3-trifluoromethylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;1-Benzyl-4-(R)-benzyl-2,5-imidazolidinedione-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;and1-Benzyl-4-(R)-benzyl-2,5-imidazolidinedione-N-[1-(3-(5-(3-trifluoromethy-1benzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide amongothers.

Likewise, U.S. Pat. No. 5,869,455 discloses N-substituted derivatives;U.S. Pat. No. 5,861,380 protease inhibitors-keto and di-keto containingring systems; U.S. Pat. No. 5,807,829 serine proteaseinhibitor-tripeptoid analogues; U.S. Pat. No. 5,801,148 serine proteaseinhibitors-proline analogues; U.S. Pat. No. 5,618,792 substitutedheterocyclic compounds useful as inhibitors of serine proteases. Thesepatents and PCT publications and others as listed infra are incorporatedherein, in their entirety, by reference. Other equally advantageousmolecules, which may be used instead of α1-antitrypsin or in combinationwith α1-antitrypsin are contemplated such as in WO 98/20034 disclosingserine protease inhibitors from fleas. Without limiting to this singlereference one skilled in the art can easily and without undueexperimentation adopt compounds such as in WO98/23565 which disclosesaminoguanidine and alkoxyguanidine compounds useful for inhibitingserine proteases; WO98/50342 discloses bis-aminomethylcarbonyl compoundsuseful for treating cysteine and serine protease disorders; WO98/50420cyclic and other amino acid derivatives useful for thrombin-relateddiseases; WO 97/21690 D-amino acid containing derivatives; WO 97/10231ketomethylene group-containing inhibitors of serine and cysteineproteases; WO 97/03679 phosphorous containing inhibitors of serine andcysteine proteases; WO 98/21186 benzothiazo and related heterocyclicinhibitors of serine proteases; WO 98/22619 discloses a combination ofinhibitors binding to P site of serine proteases with chelating site ofdivalent cations; WO 98/22098 a composition which inhibits conversion ofpro-enzyme CPP32 subfamily including caspase 3 (CPP32/Yama/Apopain); WO97/48706 pyrrolo-pyrazine-diones; WO 97/33996 human placental bikunin(recombinant) as serine protease inhibitor; WO 98/46597 complex aminoacid containing molecule for treating viral infections and conditionsdisclosed hereinabove.

Other compounds having serine protease inhibitory activity are equallysuitable and effective for use in the methods of the present invention,including but not limited to: tetrazole derivatives as disclosed in WO97/24339; guanidinobenzoic acid derivatives as disclosed in WO 97/37969and in a number of U.S. Pat. Nos. 4,283,418; 4,843,094; 4,310,533;4,283,418; 4,224,342; 4,021,472; 5,376,655; 5,247,084; and 5,077,428;phenylsulfonylamide derivatives represented by general formula in WO97/45402; novel sulfide, sulfoxide and sulfone derivatives representedby general formula in WO 97/49679; novel amidino derivatives representedby general formula in WO 99/41231; other amidinophenol derivatives asdisclosed in U.S. Pat. Nos. 5,432,178; 5,622,984; 5,614,555; 5,514,713;5,110,602; 5,004,612; and 4,889,723 among many others.

Mycobacterial Diseases Addressed by the Invention

Specific mycobacterial diseases or disorders for which the therapeuticmethods of inhibiting the mycobacterial infection of macrophages of theinvention are beneficial include, but are not limited to, thosemycobacterial diseases or disorders caused by mycobacteria from thegenus Mycobacterium that includes M. tuberculosis, M. bovis, M. leprae,M. avium-intracellulare, M. chelonei (also known as borstelense andabscessus), M. africanum, M. marinium (also known as balnei andplatypoecilus), M. buruli (also known as ulcerans), M. fortuitum (alsoknown as giae, minetti, and ranae), M. haemophilum, M. intracellulare,M. kansasii (also known as luciflavum), M. littorale (also known asxenopi), M. malmoense, M. marianum (also known as scrofulaceum andparaffinicum), M. simiae, M. szulgai, M. ulcerans, M. avium (also knownas brunense), M. flavascens, M. lepraemurium, M. microti, and M.paratuberculosis (which is the causative agent for Johne's Disease, anda possible cause of Crohn's disease), M. gordonae (also known as aquae),M. gastri, M. phlei (also known as moelleri and as timothy bacillus), M.nonchromogenicum, M. smegmatis, M. terrae, M. triviale, and M. vaccae.

In another embodiment, the Mycobacterium inhibited from infectingmacrophages comprises a Mycobacterium from the genus Mycobacterium thatincludes non-tuberculosis mycobacteria that are divided into four groupscomprising Runyon groups, selected from the group consisting of Group I(slow-growing photochromogens), Group II (slow-growing scotochromogens),Group III (slow-growing nonphotochromogens), and Group IV(rapidly-growing mycobacteria).

Bacillus anthracis and Anthrax Toxin

Anthrax toxin, produced by the gram positive rod-shaped aerobic,spore-forming bacterium Bacillus anthracis, is the toxic virulencefactor secreted by this organism. B. anthracis is often considered foruse as a biological weapon due to the potency of the secreted exotoxin,and to the capacity of the bacterium to form dormant spores which resistharsh environmental conditions. Sporulation enables ready transport anddistribution of large quantities of toxin-producing bacteria. The toxinis actually a composite consisting of 3 separate secreted proteins fromthe bacterium. The 3 proteins are protective antigen (PA), lethal factor(LF), and edema factor (EF). While LF and EF directly damage cells andare thought to cause disease due to anthrax toxin exposure, the PA isthe focus of this present disclosure. PA is crucial to the virulence ofanthrax toxin, since the PA molecule is designed to import both LF andEF inside the membranes of cells. In the absence of PA-inducedintracellular transport, anthrax toxin is unable to effect tissuedestruction, since LF and EF only function from within the cell. Theimportance of PA in the function of anthrax toxin is underscored by theeffective use of PA as the immunogen in anthrax vaccine. By generatingan immune response against PA, the vaccine confers protection againstfull (3 component) anthrax toxin.

A closer examination of the interaction between PA and the host cellsattacked by anthrax toxin is instructive. PA is first secreted by B.anthracis in a large and functionally inactive form. This inactive PAbinds to a receptor on the surface of host cells. The PA receptor hasrecently been isolated and sequenced, and found to possess vonWillebrand Factor-like regions. After docking on the surface of hostcells, PA interacts with a protease present on the cell surface. Theprotease cuts (processes) the large and inactive PA molecule into asmaller and active remnant. The identity of this protease has been thefocus of scant research effort, and it is poorly characterized. However,prior studies have shown that the protease has characteristics thatsuggest it is a host-derived serine protease. A possible serine proteasecandidate noted in the literature is furin (itself a serine protease),but other serine proteases, such as elastase, proteinase-3, or trypsinare possible alternatives. Once processed by the action of thecell-surface serine protease(s), the activated PA moleculesself-assemble into groups of 7 (heptamers) on the cell surface. Theseheptames function as the transport vehicle to deliver LF and EF insideof the cell. Once inside the cell, LF and EF initiate abnormalities incell function.

A novel approach to nullify the action of anthrax toxin is to blockaccess of the toxin to the interior of the cell. The present inventorhas shown, in previous extensive laboratory studies (Leland Shapiro etal. Facet 2000 vol. 15: 115-122, and unpublished data of Dr. LelandShapiro), that serine proteases residing on the cell surface can beneutralized by the action of several types of molecules which inhibitserine protease function. The most important natural, endogenousinhibitor of serine proteases is α-1-antitrypsin (AAT). It is noteworthythat AAT levels are reduced in lymphatics, and that anthrax toxinproduction and disease manifestations originate from within thelymphatics. It is possible that toxin production occurs in lymphatictissues because the reduced amounts of AAT provide a microenvironmentconducive to enhanced serine protease function. Such conditions areexpected to augment production of activated anthrax toxin. Therefore,administering to the subject a pharmaceutically effective amount of asubstance exhibiting mammalian α1-antitrypsin or inhibitor of serineprotease activity serves to attenuate or abolish the activity of anthraxtoxin by blocking the activity of the host-derived serine protease thatresides on the cell surface. This will negate the cell-surfaceprocessing of inactive large PA into the active smaller PA remnant.Thus, by interfering with the host-derived serine protease's activity,this will disrupt the ability of heptameric PA63 to form the prepore andultimately the pore. By disarming the anthrax toxin using this novelapproach (See FIGS. 4A-4H) several advantages are obtained compared toalternative approaches, for example, and not by way of limitation:

1. Serine protease inhibition, as a strategy to treat anthrax infection,is highly likely to be impervious to bacterial mutation due to selectivepressure. By choosing to target or inhibit the serine proteases of hostcell origin, the target molecule is immutable.

2. Synthetic inhibitors of serine proteases (AAT-like mimics) can andhave been developed (See, infra, CE-2072). Such a pharmaceutical agentmay be formulated into a pill for oral consumption in the field orformulated as an inhaler to treat inhalation anthrax.

3. Commercially available agents already approved for alternate use inhumans will work as a treatment for anthrax. These agents are currentlyused for indications other than anthrax toxicity, and include injectableAAT, plasma preparations, aprotinin and others (American J. Of RespCritical Care Med 1998, VII 158: 49-59). One possible instantiation ofthis invention may be of immediate practical application. Inhibitors ofserine proteases have been delivered to patients by inhalation. Sincethe most lethal form of anthrax infection is pulmonary invasion, aninhaled agent (natural AAT or a synthetic AAT-like mimic/or otherinhibitor of serine protease) may be especially useful due to elevatedlocal concentrations, ease of drug delivery, and lack of side effects(since administration is not systemic). This mode of focused drugdelivery may augment serine protease inhibitor activity within thepulmonary and mediastinal lymphatics, which are the principle siteswhere anthrax is thought to initiate fulminant disease.

4. By neutralizing the anthrax toxin, the direct cause of disease isdisrupted in infected individuals. Antibiotics, on the other hand, donot target toxin activity, and cannot affect toxin produced prior todestruction of the bacteria. This invention specifically contemplatesinhibiting host cell serine proteases in conjunction with administrationof one or more anti-bacterial antibiotics. Antibiotics will stop furthertoxin production by preventing the growth of bacteria and/or killing thebacterial source of toxin.

5. This approach to anthrax therapy is likely to be safe. There is anextensive clinical experience using injectable AAT to treat patientswith genetic AAT deficiency. No long-term untoward effects have beendetected to date (American J. Of Resp Critical Care Med 1998, VII 158:49-59; Wencker et al. Chest 2001 119:737-744). Moreover, a smallmolecule inhibitor of host serine protease has been administered topatients with Kawasaki's Disease (Ulinastatin® (urinary trypsininhibitor), Ono Pharmaceuticals), with an excellent safety andtolerability record. In addition, inhibition of host serine proteases totreat anthrax infection will only require a short treatment course, thusminimizing any potential concerns with long term exposure to AAT orAAT-like mimics/or other inhibitors of serine protease.

6. Soluble anthrax receptors (Bradley et al. Nature 2001 vol. 414),bacteriophage lysis of anthrax organisms (Schuch et al. Nature 2002 vol.418 884-889), dominant negative mutant anthrax toxin components (Sellmanet al. Science 2001 VI. 292: 695-697) and polyvalent inhibitors (Mourezet al. Nature Biotech 2001 Vol. 19,:958-961) may also be used inconjunction with the anthrax-based methods of the present invention.

Thus, in view of the above, the present invention provides methods forpreventing a symptom of anthrax in a subject suspected of having beenexposed to or thought to be at risk for exposure to Bacillus anthraciscomprising administering to the subject a pharmaceutically effectiveamount of a substance exhibiting mammalian α1-antitrypsin or inhibitorof serine protease activity. The present invention also provides amethod for ameliorating a symptom of anthrax in a subject in need ofsaid amelioration comprising administering to the subject apharmaceutically effective amount of a substance exhibiting mammalianα1-antitrypsin or inhibitor of serine protease activity.

In each of the above-recited methods, the clinical symptoms of anthraxcan be inhibited or prevented by administration of a substanceexhibiting mammalian α1-antitrypsin or inhibitor of serine proteaseactivity.

Clinical Symptoms of Anthrax

Anthrax occurs as three general clinical entities: i) inhalation, ii)cutaneous, and iii) gastrointestinal forms.

i) Inhalation anthrax is the deadliest form of the disease, and it isthe one most likely to be involved in a bioweapons altercation oraccident. Usually, an infected person inhales anthrax sporesserendipitously, or during a bioweapons attack. Following a 1-6 dayincubation period, a biphasic illness ensues. Initially, there isnon-specific malaise/fever/dry cough/myalgias, and chest pains. Thesecond phase occurs 2-3 days after the first phase, and consists ofprogression of the constitutional non-specific findings listed above, anaddition to ventilatory compromise, sweating, widening of themediastimum on radiographic studies, and edema of the neck and chest.This stage of illness is characterized by a necrotizing mediastinallymphadenitis. This second stage of disease can rapidly progress toshock and death within 2 days, and mortality rates of up to 80% havebeen reported. The mechanism of death in animal models appears to beenhanced production of pro-inflammatory cytokines, especially IL-1. Itis of note, referable to the instant invention disclosure, that lymphtissue is deficient in serine protease inhibitor activity compared toother body tissues. The implication is that anthrax toxin is selectivelyactivated in regions of the body (lymphatics) where there is animbalance in serine protease/anti-serine protease function that favorsserine protease activity. A preferred embodiment for using the instantinvention to treat inhalation anthrax is to deliver large amounts of aserine protease inhibitor (natural or synthetic) by inhalation. Thiswill result in a shift in the serine protease/serine protease inhibitorbalance in pulmonary and mediastinal lymphatic tissues towardantiprotease activity. This will result in blockade of the cell-surfaceprocessing event that is required for activity of anthrax toxin.

ii) Cutaneous anthrax is the commonest form (>95%) of anthrax infectionin humans. Upon exposure to anthrax spores, regions of denuded skin(cuts, abrasions, etc.), present an environment that enables anthraxorganisms to emerge from the spore state, to grow and replicate, andproduce anthrax toxin. Within 1 week, the area of anthrax inoculationdevelops a painless papule. Vesicles then form on or near the papuleover the ensuing 1-2 days, followed shortly by development of fever andmalaise, and a non-pitting edema surrounding the skin lesion that is dueto toxin activity. The original lesion (often now a vesicle) ruptures toform necrotic ulceration and enlargement—this results in formation ofthe eschar that characterizes cutaneous anthrax infection. In theabsence of therapy, this disease carries a 20% morality. For those whorecover, the eschar sloughs off in 1-2 weeks. A preferred embodiment ofthe instant invention for the treatment of cutaneous anthrax is toadminister a serine protease inhibitor (natural or synthetic) in atopical/cream preparation. Parenteral serine protease inhibitor therapycan also be co-administered in the event that systemic symptoms emerge,or such parenteral therapy can be administered prophylactically foranthrax that appears clinically to be localized to the skin.

iii) Gastrointestinal anthrax appears after ingestion of anthrax spores.After 2-5 days, one develops nausea/vomiting/fever, and abdominal pain.Bloody diarrhea rapidly ensues, and an “acute abdomen” manifests. Thepathology within the abdomen includes mucosal ulcerations. Also,hemorrhagic mesenteric lymphadenitis develops, and this is againconsistent with selective activation of the anthrax toxin in serineprotease-inhibitor deficient microenvironments. This disease carries amortality rate of 50%.

Isolated Proteins for Use in the Compositions and Methods of theInvention

One aspect of the invention pertains to isolated proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise antibodies directed against apolypeptide of the invention. In one embodiment, the native polypeptidecan be isolated from cells or tissue sources by an appropriatepurification scheme using standard protein purification techniques. Inanother embodiment, polypeptides of the invention are produced byrecombinant DNA techniques. Alternative to recombinant expression, apolypeptide of the invention can be synthesized chemically usingstandard peptide synthesis techniques.

Recombinant unmodified and mutant variants of ca.sub.1-antitrypsinproduced by genetic engineering methods are also known (U.S. Pat. No.4,711,848). The nucleotide sequence of human α1-antitrypsin and otherhuman α1-antitrypsin variants has been disclosed in internationalpublished application No. WO 86/00,337, the entire contents of which areincorporated herein by reference. This nucleotide sequence may be usedas starting material to generate all of the AAT amino acid variants andamino acid fragments depicted herein, using recombinant DNA techniquesand methods known to those of skill in the art.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”). When the protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When the protein isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the proteinhave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest.

Biologically active portions of a polypeptide of the invention includepolypeptides comprising amino acid sequences sufficiently identical toor derived from the amino acid sequence of the protein (e.g., the aminoacid sequence shown in any of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7; 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, which includefewer amino acids than the full length protein, and exhibit at least oneactivity of the corresponding full-length protein. Typically,biologically active portions comprise a domain or motif with at leastone activity of the corresponding protein. A biologically active portionof a protein of the invention can be a polypeptide which is, forexample, 10, 25, 50, 100 or more amino acids in length. Moreover, otherbiologically active portions, in which other regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated for oneor more of the functional activities of the native form of a polypeptideof the invention.

Preferred polypeptides have the amino acid sequence of SEQ ID NOs: 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, and 60. Other useful proteins are substantially identical (e.g., atleast about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%) to any ofSEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, and 60, and retain the functional activity ofthe protein of the corresponding naturally-occurring protein yet differin amino acid sequence due to natural allelic variation or mutagenesis.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment, the two sequences are the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. 90:5873-5877. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol.Biol. 215:403-410. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can beused to perform an iterated search which detects distant relationshipsbetween molecules (Id.). When utilizing BLAST, Gapped BLAST, andPSI-Blast programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used.

Another preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS (1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) which is part of the CGC sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 can be used. Additional algorithms forsequence analysis are known in the art and include ADVANCE and ADAM asdescribed in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5;and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci.85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search. If ktup=2, similar regions in thetwo sequences being compared are found by looking at pairs of alignedresidues; if ktup=1, single aligned amino acids are examined. ktup canbe set to 2 or 1 for protein sequences, or from 1 to 6 for DNAsequences. The default if ktup is not specified is 2 for proteins and 6for DNA. For a further description of FASTA parameters, seehttp://bioweb.pasteur.fr/d-ocs/Man/man/fasta.1.html #sect2, the contentsof which are incorporated herein by reference.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

The present invention also pertains to variants of the polypeptides ofthe invention. Such variants have an altered amino acid sequence whichcan function as either agonists (mimetics) or as antagonists. Variantscan be generated by mutagenesis, e.g., discrete point mutation ortruncation. An agonist can retain substantially the same, or a subset,of the biological activities of the naturally occurring form of theprotein. An antagonist of a protein can inhibit one or more of theactivities of the naturally occurring form of the protein by, forexample, competitively binding to a downstream or upstream member of acellular signaling cascade which includes the protein of interest. Thus,specific biological effects can be elicited by treatment with a variantof limited function. Treatment of a subject with a variant having asubset of the biological activities of the naturally occurring form ofthe protein can have fewer side effects in a subject relative totreatment with the naturally occurring form of the protein.

Variants of a protein of the invention which function as either agonists(mimetics) or as antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theprotein of the invention for agonist or antagonist activity. In oneembodiment, a variegated library of variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential protein sequences is expressible as individual polypeptides,or alternatively, as a set of larger fusion proteins (e.g., for phasedisplay). There are a variety of methods which can be used to producelibraries of potential variants of the polypeptides of the inventionfrom a degenerate oligonucleotide sequence. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang(1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the coding sequence of apolypeptide of the invention can be used to generate a variegatedpopulation of polypeptides for screening and subsequent selection ofvariants. For example, a library of coding sequence fragments can begenerated by treating a double stranded PCR fragment of the codingsequence of interest with a nuclease under conditions wherein nickingoccurs only about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with S1 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal and internal fragments of various sizes of the protein ofinterest.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofa protein of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

An isolated polypeptide of the invention, or a fragment thereof, can beused as an immunogen to generate antibodies using standard techniquesfor polyclonal and monoclonal antibody preparation. The full-lengthpolypeptide or protein can be used or, alternatively, the inventionprovides antigenic peptide fragments for use as immunogens. Theantigenic peptide of a protein of the invention comprises at least 8(preferably 10, 15, 20, or 30) amino acid residues of the amino acidsequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, and 60, and encompasses an epitopeof the protein such that an antibody raised against the peptide forms aspecific immune complex with the protein.

Fusion Proteins for Use in the Compositions and Methods of the Invention

In each of the aforementioned aspects and embodiments of the invention,fusion polypeptides are also specifically contemplated herein.

In one embodiment, fusion polypeptides of the invention are produced byrecombinant DNA techniques. Alternative to recombinant expression, afusion polypeptide of the invention can be synthesized chemically usingstandard peptide synthesis techniques. The present invention alsoprovides compositions that comprise a fusion polypeptide of theinvention and a pharmaceutically acceptable carrier, excipient ordiluent.

In each of the above-recited methods, the mammalian α1-antitrypsin orinhibitor of serine protease activity substance may be part of a fusionpolypeptide, wherein said fusion polypeptide comprises mammalianα1-antitrypsin or inhibitor of serine protease activity substance and anamino acid sequence heterologous to said mammalian α1-antitrypsin orinhibitor of serine protease activity substance.

Among the particular fusion polypeptides of the invention are, forexample, fusion polypeptides that comprise the amino acid sequence ofthe α1-antitrypsin depicted below in SEQ ID NO:63.

1         01         01         01         01        0MPSSVSWGIL LAGLCCLVPV SLAEDPQGDA AQKTDTSHHD QDHPTFNKITPNLAEFAFSL YRQLAHQSNS TNIFFSPVSI ATAFANLSLG TKADTHDEIL 100EGLNFNLTEI PEAQIHEGFQ ELLRTLNQPD SQLQLTTGNG LFLSEGLKLVDKFLEDVKKL YHSEAFTVNF GDHEEAKKQI NDYVEKGTQG KIVDLVKELD 200RDTVFALVNY IFFKGKWERP FEVKDTEDED HVDQVTTVK VPMMKRLGMFNIQHCKKLSS WVLLMKYLGN ATAIFFLPDE GKLQHLENEL THDIITKFLE 300NEDRRSASLH LPKLSITGTY DLKSVLGQLG ITKVFSNGAD LSGVTEEAPLKLSKAVHKAV LTIDEKGTEA AGAMFLEAIP MSIPPEVKFN KPFVFLMIEQ 400NTKSPLFMGK VVNPTQK 417

The fusion polypeptides of the invention can be such that theheterologous amino acid sequence comprises a human immunoglobulinconstant region, such as a human IgG1 constant region, including amodified human IgG1 constant region wherein the IgG1 constant regiondoes not bind Fc receptor and/or does not initiate antibody-dependentcellular cytotoxicity (ADCC) reactions.

In particular, in one embodiment the fusion protein comprises aheterologous sequence that is a sequence derived from a member of theimmunoglobulin protein family, for example, comprise an immunoglobulinconstant region, e.g., a human immunoglobulin constant region such as ahuman IgG1 constant region. The fusion protein can, for example,comprise a portion of a mammalian α1-antitrypsin or inhibitor of serineprotease activity polypeptide fused with the amino-terminus or thecarboxyl-terminus of an immunoglobulin constant region, as disclosed,e.g., in U.S. Pat. Nos. 5,714,147, 5,116,964, 5,514,582, and 5,455,165.In those embodiments in which all or part of a polypeptide of theinvention is fused with sequences derived from a member of theimmunoglobulin protein family, the FcR region of the immunoglobulin maybe either wild-type or mutated. In certain embodiments, it is desirableto utilize an immunoglobulin fusion protein that does not interact witha Fc receptor and does not initiate ADCC reactions. In such instances,the immunoglobulin heterologous sequence of the fusion protein can bemutated to inhibit such reactions. See, e.g. U.S. Pat. No. 5,985,279 andWO 98/06248.

The heterologous amino acid sequence of the fusion polypeptides utilizedas part of the present invention can also comprise an amino acidsequence useful for identifying, tracking or purifying the fusionpolypeptide, e.g., can comprise a FLAG or a His tag sequence. The fusionpolypeptide can further comprise an amino acid sequence containing aproteolytic cleavage site which can, for example, be useful for removingthe heterologous amino acid sequence from the α1-antitrypsin orinhibitor of serine protease derivative or mimic sequence of the fusionpolypeptide.

In particular, the heterologous amino acid sequence of the fusionpolypeptides of the present invention can also comprise an amino acidsequence useful for identifying, tracking or purifying the fusionpolypeptide, e.g., can comprise a FLAG (see, e.g., Hoop, T. P. et al.,Bio/Technology 6, 1204-1210 (1988); Prickett, K. S. et al.,BioTechniques 7, 580-589 (1989)) or a His tag (Van Reeth, T. et al.,BioTechniques 25, 898-904 (1998)) sequence. The fusion polypeptide canfurther comprise an amino acid sequence containing a proteolyticcleavage site which can, for example, be useful for removing theheterologous amino acid sequence from the mammalian α1-antitrypsin orinhibitor of serine protease activity polypeptide sequence of the fusionpolypeptide.

In yet another embodiment, the mammalian α1-antitrypsin or inhibitor ofserine protease-like activity polypeptide fusion protein comprises a GSTfusion protein in which the mammalian α1-antitrypsin or inhibitor ofserine protease activity polypeptide of the invention is fused to theC-terminus of GST sequences. Such a fusion protein can facilitate thepurification of a recombinant polypeptide of the invention. In thoseembodiments in which a GST, FLAG or HisTag fusion constructs is employedin the construction of the mammalian α1-antitrypsin or inhibitor ofserine protease activity polypeptide fusion proteins, proteolyticcleavage sites may be optionally introduced at the junction of thefusion moiety and the mammalian α1-antitrypsin or inhibitor of serineprotease activity polypeptide to enable separation of the mammalianα1-antitrypsin or inhibitor of serine protease activity polypeptide fromthe fusion moiety subsequent to purification of the mammalianα1-antitrypsin or inhibitor of serine protease activity polypeptide.Such enzymes, and their cognate recognition sequences, include, forexample, without limitation, Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc.;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which may beused to fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target mammalian α1-antitrypsin orinhibitor of serine protease activity polypeptide protein.

Expression vectors can routinely be designed for expression of a fusionpolypeptide of the invention in prokaryotic (e.g., E. coli) oreukaryotic cells (e.g., insect cells (using baculovirus expressionvectors), yeast cells or mammalian cells). Suitable host cells arediscussed further in Goeddel, supra. Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET lid (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET lid vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a residentprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacterium with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSecl (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpPicZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, the expression vector is a baculovirus expression vector.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal., supra.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the alpha-fetoprotein promoter (Campesand Tilghman (1989) Genes Dev. 3:537-546).

A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell(e.g., insect cells, yeast or mammalian cells).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra), and other laboratorymanuals.

Combination Therapies for Treating Mycobacterial Diseases and AnthraxUsing the Methods of the Invention

In each of the aforementioned aspects and embodiments of the invention,combination therapies other than those enumerated above are alsospecifically contemplated herein. In particular, the compositions of thepresent invention may be administered with one or more macrolide ornon-macrolide antibiotics, anti-bacterial agents, anti-fungicides,anti-viral agents, and anti-parasitic agents, anti-inflammatory orimmunomodulatory drugs or agents.

Examples of macrolide antibiotics that may be used in combination withthe composition of the present invention include, inter alia, thefollowing synthetic, semi-synthetic or naturally occurring microlidicantibiotic compounds: methymycin, neomethymycin, YC-17, litorin,erythromycin A to F, oleandomycin, roxithromycin, dirithromycin,flurithromycin, clarithromycin, davercin, azithromycin, josamycin,kitasamycin, spiramycin, midecamycin, rokitamycin, miokamycin,lankacidin, and the derivatives of these compounds. Thus, erythromycinand compounds derived from erythromycin belong to the general class ofantibiotics known as “macrolides.” Examples of preferred erythromycinand erythromycin-like compounds include: erythromycin, clarithromycin,azithromycin, and troleandomycin.

Additional antibiotics, other than the macrolidic antibiotics describedabove, which are suitable for use in the methods of the presentinvention include, for example, any molecule that tends to prevent,inhibit or destroy life and as such, and as used herein, includesanti-bacterial agents, anti-fungicides, anti-viral agents, andanti-parasitic agents. These agents may be isolated from an organismthat produces the agent or procured from a commercial source (e.g.,pharmaceutical company, such as Eli Lilly, Indianapolis, Ind.; Sigma,St. Louis, Mo.).

For example, the anti-TB antibiotic isoniazid (isonicotinic acidhydrazide) is frequently effective, but isoniazid often causes severe,sometimes fatal, hepatitis. The risk of hepatitis increases with thepatient's age. Additionally, isoniazid causes peripheral neuropathy insome recipients in a dose-related fashion. Rifampin, another antibioticused to treat TB, must be used in conjunction with another drug such asisoniazid. This requirement for combination therapy with rifampinapplies to the initial treatment as well as the retreatment of pulmonaryTB.

Usually, isoniazid, rifampin, ethambutol and ethionamide are givenorally. Streptomycin is typically given intramuscularly. Amikacin isgiven intramuscularly or intravenously. Clofazimine, which is also usedto treat leprosy, is given orally.

Amikacin is a semisynthetic aminoglycoside antibiotic derived fromKanamycin A. For its preparation see U.S. Pat. No. 3,781,268. For areview see Kerridge, Pharmacological and Biochemical Properties of DrugSubstances 1:125-153, M. E. Goldberg, ed. (1977). Amikacin is usuallyadministered intramuscularly or intravenously. For additionalinformation including clinical pharmacology, indications, side effectsand dosages, see the Physicians' Desk Reference, 42 ed. (1988) at pages744-746 (hereinafter, PDR).

Clofazimine is an antibacterial agent also known as LAMPRENE® For itspreparation, see Barry, et al., Nature 179:1013 (1957). For a review seeKarat, et al., Brit. Med. J. 3:175 (1971). Clofazimine is generallygiven orally. For additional information including clinicalpharmacology, precautions and dosages, see the PDR at page 982.

Ethionamide is an antibacterial agent also known as AMIDAZINE® andTRECATOR® See British Patent No. 800,250. This drug is typically givenorally. For further information including precautions and dosages, seethe PDR at page 2310.

Ciprofloxacin is a broad spectrum synthetic antibacterial agent for oralusage. It is also known as CIPRO® It is typically given in total dailydosages of 500 to 1,000 milligrams which is usually given in 2 equaldoses in 24 hours. For further information see the PDR (1989) at pages1441-1443. other member of this fluoroquinolone class of antibioticsinclude ofloxacin, levofloxacin, troveofloxacin, pefloxacin,gatifloxacin, and moxifloxacin.

Other examples of anti-bacterial antibiotic agents include, but are notlimited to, penicillins, cephalosporins, carbacephems, cephamycins,carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones,tetracyclines, macrolides, oxazalidinones, and fluoroquinolones.Examples of antibiotic agents include, but are not limited to,Penicillin G (CAS Registry No.: 61-33-6); Methicillin (CAS Registry No.:61-32-5); Nafcillin (CAS Registry No.: 147-52-4); Oxacillin (CASRegistry No.: 66-79-5); Cloxacillin (CAS Registry No.: 61-72-3);Dicloxacillin (CAS Registry No.: 3116-76-5); Ampicillin (CAS RegistryNo.: 69-53-4); Amoxicillin (CAS Registry No.: 26787-78-0); Ticarcillin(CAS Registry No.: 34787-01-4); Carbenicillin (CAS Registry No.:4697-36-3); Mezlocillin (CAS Registry No.: 51481-65-3); Azlocillin (CASRegistry No.: 37091-66-0); Piperacillin (CAS Registry No.: 61477-96-1);Imipenem (CAS Registry No.: 74431-23-5); Aztreonam (CAS Registry No.:78110-38-0); Cephalothin (CAS Registry No.: 153-61-7); Cefazolin (CASRegistry No.: 25953-19-9); Cefaclor (CAS Registry No.: 70356-03-5);Cefamandole formate sodium (CAS Registry No.: 42540-40-9); Cefoxitin(CAS Registry No.: 35607-66-0); Cefuroxime (CAS Registry No.:55268-75-2); Cefonicid (CAS Registry No.: 61270-58-4); Cefinetazole (CASRegistry No.: 56796-20-4); Cefotetan (CAS Registry No.: 69712-56-7);Cefprozil (CAS Registry No.: 92665-29-7); Loracarbef (CAS Registry No.:121961-22-6); Cefetamet (CAS Registry No.: 65052-63-3); Cefoperazone(CAS Registry No.: 62893-19-0); Cefotaxime (CAS Registry No.:63527-52-6); Ceftizoxime (CAS Registry No.: 68401-81-0); Ceftriaxone(CAS Registry No.: 73384-59-5); Ceftazidime (CAS Registry No.:72558-82-8); Cefepime (CAS Registry No.: 88040-23-7); Cefixime (CASRegistry No.: 79350-37-1); Cefpodoxime (CAS Registry No.: 80210-62-4);Cefsulodin (CAS Registry No.: 62587-73-9); Fleroxacin (CAS Registry No.:79660-72-3); Nalidixic acid (CAS Registry No.: 389-08-2); Norfloxacin(CAS Registry No.: 70458-96-7); Ciprofloxacin (CAS Registry No.:85721-33-1); Ofloxacin (CAS Registry No.: 82419-36-1); Enoxacin (CASRegistry No.: 74011-58-8); Lomefloxacin (CAS Registry No.: 98079-51-7);Cinoxacin (CAS Registry No.: 28657-80-9); Doxycycline (CAS Registry No.:564-25-0); Minocycline (CAS Registry No.: 10118-90-8); Tetracycline (CASRegistry No.: 60-54-8); Amikacin (CAS Registry No.: 37517-28-5);Gentamicin (CAS Registry No.: 1403-66-3); Kanamycin (CAS Registry No.:8063-07-8); Netilmicin (CAS Registry No.: 56391-56-1); Tobramycin (CASRegistry No.: 32986-56-4); Streptomycin (CAS Registry No.: 57-92-1);Azithromycin (CAS Registry No.: 83905-01-5); Clarithromycin (CASRegistry No.: 81103-11-9); Erythromycin (CAS Registry No.: 114-07-8);Erythromycin estolate (CAS Registry No.: 3521-62-8); Erythromycin ethylsuccinate (CAS Registry No.: 41342-53-4); Erythromycin glucoheptonate(CAS Registry No.: 23067-13-2); Erythromycin lactobionate (CAS RegistryNo.: 3847-29-8); Erythromycin stearate (CAS Registry No.: 643-22-1);Vancomycin (CAS Registry No.: 1404-90-6); Teicoplanin (CAS Registry No.:61036-64-4); Chloramphenicol (CAS Registry No.: 56-75-7); Clindamycin(CAS Registry No.: 18323-44-9); Trimethoprim (CAS Registry No.:738-70-5); Sulfamethoxazole (CAS Registry No.: 723-46-6); Nitrofurantoin(CAS Registry No.: 67-20-9); Rifampin (CAS Registry No.: 13292-46-1);Mupirocin (CAS Registry No.: 12650-69-0); Metronidazole (CAS RegistryNo.: 443-48-1); Cephalexin (CAS Registry No.: 15686-71-2); Roxithromycin(CAS Registry No.: 80214-83-1); Co-amoxiclavuanate; combinations ofPiperacillin and Tazobactam; and their various salts, acids, bases, andother derivatives.

Anti-fungal agents include, but are not limited to, caspofungin,terbinafine hydrochloride, nystatin, amphotericin B, griseofulvin,ketoconazole, miconazole nitrate, flucytosine, fluconazole,itraconazole, clotrimazole, benzoic acid, salicylic acid, and seleniumsulfide.

Anti-viral agents include, but are not limited to, valgancyclovir,amantadine hydrochloride, rimantadin, acyclovir, famciclovir, foscamet,ganciclovir sodium, idoxuridine, ribavirin, sorivudine, trifluridine,valacyclovir, vidarabin, didanosine, stavudine, zalcitabine, zidovudine,interferon alpha, and edoxudine.

Anti-parasitic agents include, but are not limited to,pirethrins/piperonyl butoxide, permethrin, iodoquinol, metronidazole,diethylcarbamazine citrate, piperazine, pyrantel pamoate, mebendazole,thiabendazole, praziquantel, albendazole, proguanil, quinidine gluconateinjection, quinine sulfate, chloroquine phosphate, mefloquinehydrochloride, primaquine phosphate, atovaquone, co-trimoxazole(sulfamethoxazole/trimethoprim), and pentamidine isethionate.

In another aspect, in the method of the present invention, one may, forexample, supplement the composition by administration of atherapeutically effective amount of one or more an anti-inflammatory orimmunomodulatory drugs or agents. By “immunomodulatory drugs or agents”,it is meant, e.g., agents which act on the immune system, directly orindirectly, e.g., by stimulating or suppressing a cellular activity of acell in the immune system, e.g., T-cells, B-cells, macrophages, orantigen presenting cells (APC), or by acting upon components outside theimmune system which, in turn, stimulate, suppress, or modulate theimmune system, e.g., hormones, receptor agonists or antagonists, andneurotransmitters; immunomodulators can be, e.g., immunosuppressants orimmunostimulants. By “anti-inflammatory drugs”, it is meant, e.g.,agents which treat inflammatory responses, i.e., a tissue reaction toinjury, e.g., agents which treat the immune, vascular, or lymphaticsystems.

Anti-inflammatory or immunomodulatory drugs or agents suitable for usein this invention include, but are not limited to, interferonderivatives, e.g., betaseron, .beta.-interferon; prostane derivatives,e.g., compounds disclosed in PCT/DE93/0013, e.g., iloprost, cicaprost;glucocorticoid, e.g., cortisol, prednisolone, methylprednisolone,dexamethasone; immunsuppressives, e.g., cyclosporine A, FK-506,methoxsalene, thalidomide, sulfasalazine, azathioprine, methotrexate;lipoxygenase inhibitors, e.g., zileutone, MK-886, WY-50295, SC-45662,SC-41661A, BI-L-357; leukotriene antagonists, e.g., compounds disclosedin DE 40091171 German patent application P 42 42 390.2; WO 9201675;SC-41930; SC-50605; SC-51146; LY 255283 (D. K. Herron et al., FASEB J.2: Abstr. 4729, 1988); LY 223982 (D. M. Gapinski et al. J. Med. Chem.33: 2798-2813, 1990); U-75302 and analogs, e.g., described by J. Morriset al., Tetrahedron Lett. 29: 143-146, 1988, C. E. Burgos et al.,Tetrahedron Lett. 30: 5081-5084, 1989; B. M. Taylor et al.,Prostaglandins 42: 211-224, 1991; compounds disclosed in U.S. Pat. No.5,019,573; ONO-LB-457 and analogs, e.g., described by K. Kishikawa etal., Adv. Prostagl. Thombox. Leukotriene Res. 21: 407-410, 1990; M.Konno et al., Adv. Prostagl. Thrombox. Leukotriene Res. 21: 411-414,1990; WF-11605 and analogs, e.g., disclosed in U.S. Pat. No. 4,963,583;compounds disclosed in WO 9118601, WO 9118879; WO 9118880, WO 9118883,antiinflammatory substances, e.g., NPC 16570, NPC 17923 described by L.Noronha-Blab. et al., Gastroenterology 102 (Suppl.): A 672, 1992; NPC15669 and analogs described by R. M. Burch et al., Proc. Nat. Acad. Sci.USA 88: 355-359, 1991; S. Pou et al., Biochem. Pharmacol. 45: 2123-2127,1993; peptide derivatives, e.g., ACTH and analogs; solubleTNF-receptors; TNF-antibodies; soluble receptors of interleukines, othercytokines, T-cell-proteins; antibodies against receptors ofinterleukins, other cytokines, and T-cell-proteins.

The therapeutic agents of the instant invention may be used for thetreatment of animal subjects or patients, and more preferably, mammals,including humans, as well as mammals such as non-human primates, dogs,cats, horses, cows, pigs, guinea pigs, and rodents.

Modes of Administration

Modes of administration of the various therapeutic agents used in theinvention are exemplified below. However, the agents can be delivered byany of a variety of routes including: by injection (e.g., subcutaneous,intramuscular, intravenous, intra-arterial, intraperitoneal), bycontinuous intravenous infusion, cutaenously, dermally, transdermally,orally (e.g., tablet, pill, liquid medicine), by implanted osmotic pumps(e.g., Alza Corp.), by suppository or aerosol spray.

The peptide-based serine protease inhibitors may be prepared by anysuitable synthesis method such as originally described by Merrifield, J.Am. Chem. Soc., 85, p 2149 (1963). Synthetic peptides which exhibitinhibitory activity toward serine proteases and methods for preparingand using same are disclosed for example in U.S. Pat. Nos. 4,829,052,5,157,019 to Glover; U.S. Pat. No. 5,420,110 to Miller; U.S. Pat. No.4,963,654 Katunuma as incorporated herein by reference.

Those skilled in the art of biochemical synthesis will recognize thatfor commercial-scale quantities of peptides, such peptides arepreferably prepared using recombinant DNA techniques, synthetictechniques, or chemical derivatization of biologically or chemicallysynthesized peptides.

The compounds of the present invention are used as therapeutic agents inthe treatment of a physiological (especially pathological) conditioncaused in whole or part, by excessive serine protease activity. Thepeptides may be administered as free peptides or pharmaceuticallyacceptable salts thereof. The terms used herein conform to those foundin Budavari, Susan (Editor), “The Merck Index” An Encyclopedia ofChemicals, Drugs, and Biologicals; Merck & Co., Inc. The term“pharmaceutically acceptable salt” refers to those acid addition saltsor metal complexes of the peptides which do not significantly oradversely affect the therapeutic properties (e.g. efficacy, toxicity,etc.) of the peptides. The peptides should be administered toindividuals as a pharmaceutical composition, which, in most cases, willcomprise the peptide and/or pharmaceutical salts thereof with apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” refers to those solid and liquid carriers, which donot significantly or adversely affect the therapeutic properties of thepeptides.

The pharmaceutical compositions containing peptides of the presentinvention may be administered to individuals, particularly humans,either intravenously, subcutaneously, intramuscularly, intranasally,orally, topically, transdermally, parenterally, gastrointestinally,transbronchially and transalveolarly. Topical administration isaccomplished via a topically applied cream, gel, rinse, etc. containingtherapeutically effective amounts of inhibitors of serine proteases.Transdermal administration is accomplished by application of a cream,rinse, gel, etc. capable of allowing the inhibitors of serine proteasesto penetrate the skin and enter the blood stream. Parenteral routes ofadministration include, but are not limited to, direct injection such asintravenous, intramuscular, intraperitoneal or subcutaneous injection.Gastrointestinal routes of administration include, but are not limitedto, ingestion and rectal. Transbronchial and transalveolar routes ofadministration include, but are not limited to, inhalation, either viathe mouth or intranasally and direct injection into an airway, such asthrough a tracheotomy, tracheostomy, endotracheal tube, or metered doseor continuous inhaler. In addition, osmotic pumps may be used foradministration. The necessary dosage will vary with the particularcondition being treated, method of administration and rate of clearanceof the molecule from the body.

Although the compounds described herein and/or their derivatives may beadministered as the pure chemicals, it is preferable to present theactive ingredient as a pharmaceutical composition. The invention thusfurther provides the use of a pharmaceutical composition comprising oneor more compounds and/or a pharmaceutically acceptable salt thereof,together with one or more pharmaceutically acceptable carriers thereforeand, optionally, other therapeutic and/or prophylactic ingredients. Thecarrier(s) must be acceptable in the sense of being compatible with theother ingredients of the composition and not deleterious to therecipient thereof.

Pharmaceutical compositions include those suitable for oral orparenteral (including intramuscular, subcutaneous, cutaneous, inhaledand intravenous) administration. The compositions may, whereappropriate, be conveniently presented in discrete unit dosage forms andmay be prepared by any of the methods well known in the art of pharmacy.Such methods include the step of bringing into association the activecompound with liquid carriers, solid matrices, semi-solid carriers,finely divided solid carriers or combinations thereof, and then, ifnecessary, shaping the product into the desired delivery system.

Pharmaceutical compositions suitable for oral administration may bepresented as discrete unit dosage forms such as hard or soft gelatincapsules, cachets or tablets, each containing a predetermined amount ofthe active ingredient; as a powder or as granules; as a solution, asuspension or as an emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. Tablets and capsules for oraladministration may contain conventional excipients such as bindingagents, fillers, lubricants, disintegrants, or wetting agents. Thetablets may be coated according to methods well known in the art., e.g.,with enteric coatings.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspension, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for constitution with water or anothersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, emulsifying agents,non-aqueous vehicles (which may include edible oils), or preservative.The compounds may also be formulated for parenteral administration(e.g., by injection, for example, bolus injection or continuousinfusion) and may be presented in unit dose form in ampoules, pre-filledsyringes, small bolus infusion containers or in multi-dose containerswith an added preservative. The compositions may take such forms assuspensions, solutions, or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Alternatively, the active ingredient may be in powderform, obtained by aseptic isolation of sterile solid or bylyophilization from solution, for constitution with a suitable vehicle,e.g., sterile, pyrogen-free water, before use.

For topical administration to the epidermis, the compounds may beformulated as ointments, creams or lotions, or as the active ingredientof a transdermal patch. Suitable transdermal delivery systems aredisclosed, for example, in Fisher et al. (U.S. Pat. No. 4,788,603) orBawas et al. (U.S. Pat. Nos. 4,931,279, 4,668,504 and 4,713,224).Ointments and creams may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions may be formulated with an aqueous or oily base and willin general also contain one or more emulsifying agents, stabilizingagents, dispersing agents, suspending agents, thickening agents, orcoloring agents. The active ingredient can also be delivered viaiontophoresis, e.g., as disclosed in U.S. Pat. Nos. 4,140,122,4,383,529, or 4,051,842. At least two types of release are possible inthese systems. Release by diffusion occurs when the matrix isnon-porous. The pharmaceutically effective compound dissolves in anddiffuses through the matrix itself. Release by microporous flow occurswhen the pharmaceutically effective compound is transported through aliquid phase in the pores of the matrix.

Compositions suitable for topical administration in the mouth includeunit dosage forms such as lozenges comprising active ingredient in aflavored base, usually sucrose and acacia or tragacanth; pastillescomprising the active ingredient in an inert base such as gelatin andglycerin or sucrose and acacia; mucoadherent gels, and mouthwashescomprising the active ingredient in a suitable liquid carrier.

When desired, the above-described compositions can be adapted to providesustained release of the active ingredient employed, e.g., bycombination thereof with certain hydrophilic polymer matrices, e.g.,comprising natural gels, synthetic polymer gels or mixtures thereof.

The pharmaceutical compositions according to the invention may alsocontain other adjuvants such as flavorings, coloring, antimicrobialagents, or preservatives.

It will be further appreciated that the amount of the compound, or anactive salt or derivative thereof, required for use in treatment willvary not only with the particular salt selected but also with the routeof administration, the nature of the condition being treated and the ageand condition of the patient and will be selected, ultimately, at thediscretion of the attending physician.

A pharmaceutical composition of the invention contains an appropriatepharmaceutically acceptable carrier as defined supra. These compositionscan take the form of solutions, suspensions, tablets, pills, capsules,powders, sustained-release formulations and the like. Suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences 1990, pp. 1519-1675, Gennaro, A. R., ed., Mack PublishingCompany, Easton, Pa. The serine protease inhibitor molecules of theinvention can be administered in liposomes or polymers (see, Langer, R.Nature 1998, 392, 5). Such compositions will contain an effectivetherapeutic amount of the active compound together with a suitableamount of carrier so as to provide the form for proper administration tothe subject.

In general, the compound is conveniently administered in unit dosageform; for example, containing 5 to 2000 mg, conveniently 10 to 1000 mg,most conveniently, 50 to 500 mg of active ingredient per unit dosageform.

Desirable blood levels may be maintained by continuous infusion toprovide about 0.01-5.0 mg/kg/hr or by intermittent infusions containingabout 0.4-20 mg/kg of the active ingredient(s). Buffers, preservatives,antioxidants and the like can be incorporated as required.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations, such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

Actual dosage levels of active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active compound(s) that is effective to achieve the desiredtherapeutic response for a particular patient, compositions, and mode ofadministration. The selected dosage level will depend upon the activityof the particular pharmaceutical compound or analogue thereof of thepresent invention, the route of administration, the severity of thecondition being treated, and the condition and prior medical history ofthe patient being treated. However, it is within the skill of the art tostart doses of the pharmaceutical compound at levels lower than requiredto achieve the desired therapeutic effect and to gradually increase thedosage until the desired effect is achieved.

The pharmaceutical compositions of the present invention can be used inboth veterinary medicine and human therapy. The magnitude of aprophylactic or therapeutic dose of the pharmaceutical composition ofthe invention in the acute or chronic management of pain associated withabove-mentioned diseases or indications will vary with the severity ofthe condition to be treated and the route of administration. The dose,and perhaps the dose frequency, will also vary according to the age,body weight, and response of the individual patient. In general, thetotal daily dose range of the pharmaceutical composition of thisinvention is generally between about 1 to about 100 mg, preferably about1 to about 20 mg, and more preferably about 1 to about 10 mg of activecompound per kilogram of body weight per day are administered to amammalian patient. If desired, the effective daily dose may be dividedinto multiple doses for purposes of administration, e.g. two to fourseparate doses per day.

Alternatively, the total daily dose range of the active ingredient ofthis invention should be sufficient to increase the serum concentrationof the protease inhibitor by 10-100 micromolar.

It is intended herein that by recitation of such specified ranges, theranges cited also include all those dose range amounts between therecited range. For example, in the range about 1 and 100, it is intendedto encompass 2 to 99, 3-98, etc., without actually reciting eachspecific range. The actual preferred amounts of the active ingredientwill vary with each case, according to the species of mammal, the natureand severity of the particular affliction being treated, and the methodof administration.

It is also understood that doses within those ranges, but not explicitlystated, such as 30 mg, 50 mg, 75 mg, etc. are encompassed by the statedranges, as are amounts slightly outside the stated range limits.

The actual preferred amounts of the active ingredient will vary witheach case, according to the species of mammal, the nature and severityof the particular affliction being treated, and the method ofadministration.

In general, the pharmaceutical compositions of the present invention areperiodically administered to an individual patient as necessary toimprove symptoms of the particular disease being treated. The length oftime during which the compositions are administered and the total dosagewill necessarily vary with each case, according to the nature andseverity of the particular affliction being treated and the physicalcondition of the subject or patient receiving such treatment.

It is further recommended that children, patients aged over 65 years,and those with impaired renal or hepatic function initially receive lowdoses, and that they then be titrated based on individual response(s) orblood level(s). It may be necessary to use dosages outside these rangesin some cases, as will be apparent to those of ordinary skill in theart. Further, it is noted that the clinician or treating physician willknow, with no more than routine experimentation, how and when tointerrupt, adjust, or terminate therapy in conjunction with individualpatient response.

Useful dosages of the compounds of the present invention can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known to the art; for example,see U.S. Pat. No. 4,938,949.

EXAMPLES

The following specific examples are provided to better assist the readerin the various aspects of practicing the present invention. As thesespecific examples are merely illustrative, nothing in the followingdescriptions should be construed as limiting the invention in any way.Such limitations are, or course, defined solely by the accompanyingclaims.

Example One

Effect of α1-antitrypsin on Mycobacterium Avium Complex (Mac) Infectionof Human Monocyte-Derived Macrophages

1. TB or MAC organisms were suspended at a concentration of oneMcFarland standard. One McFarland is defined as a degree of turbidity oforganisms suspended in liquid that matches that of a standard aliquot. Asample turbidity that is equivalent to that of the one McFarlandstandard represents about 10.sup.7 bacilli/ml. The optimal duration of atest culture is approximately 10-12 days of bacilli grown in Middlebrook7H9 broth (=Mycobacterium medium).

2. Infecting the cells. The cells infected were human monocyte-derivedmacrophages (MDM). MDM were isolated from human peripheral bloodmononuclear cells (PBMC) that were obtained from heparinized blood fromhealthy volunteers by centrifuging the heparinized blood over aficol-hypaque cushion. The isolated PBMC were aliquoted into polystyrenetissue culture plates and the monocytes are allowed to adhere for 2 hrs(0.5×10⁶ PBMC were added to each well, of which approximately 10-20% aremonocytes). Experiments were performed in plates without or with sterileround glass coverslips in the bottoms of the wells (see a. below). Onlythe monocytic population within the PBMC will adhere to the plates underthese conditions. The wells were then washed (to remove the non-adheringlymphocytes) and incubated in fresh medium.times.10-12 days(medium=RPMI+10% fetal calf serum+100 units/ml of penicillin G), whichallows maturation of the monocytes into macrophages. The volume ofmedium in each well was 1.0 ml. The medium was then removed from eachwell of MDM, and the wells were replenished with either medium alone(control), with AAT, or with ala-ala-pro-val-chloromethyl ketone (anAAT-like synthetic serine protease inhibitor) (Bachem, Inc.), and thewells were incubated for 3.0 hr. Then, the MDM in each well wereinfected with MAC (strain Mycobacterium avium 9141) or TB (strain H37RV)at a ratio of mycobacterial bacilli/cell of 1.times.10.sup.6. After a1.0 hr incubation (to allow the mycobacteria to bind to the MDMsurfaces), the supernatants were removed and saved for cytokine assays.The wells were then washed twice (with a 1:1 solution of RPMI andsaline),

Two independent assays were then used to quantify mycobacterialinfection of the human monocyte-derived macrophages:

a. Direct Observation and Counting of the Number of Infected Cells inEach Well

For these experiments, the mycobacteria-infected MDM were cultured inwells of a polystyrene tissue culture plate that had sterile round glasscover slips inserted into the bottoms of the wells. Since the MDMs wereoriginally seeded onto these cover slips, the MDMs adhered to the coverslip surfaces. After incubation with MAC or TB, the wells were washedtwice (as stated above) and then fixed.times.1 hr using glutaraldehyde.The mycobacteria were then stained using a mycobacterial stain(Zeihl-Nielsson) without injuring the cells. The number of infectedcells was quantified optically and the data expressed as a percent ofthe total number of MDM in each well.

b. Colony Counts

After the infected cells were washed twice (see above), the cells inparallel wells that did not contain cover slips were lysed using 1.0 mlof lysing buffer per well for 5.0 min (0.25% sDKF lysis buffer).

After the infected MDM were lysed (see above), the lysate fluid wasdiluted 1:1 with 1.0 ml of 7H9 medium. The mycobacterial suspension wasdiluted serially 1:10 into 1% (vol/vol) 7H9 medium and sterile water.The diluted mycobacterial suspensions were vortexed and then 0.5 ml ofthe suspension from each aliquot was plated onto mycobacteria medium(solid 7H9 medium). This mycobacteria-containing fluid was thencultured. The plates were incubated for 10-12 days for MAC and for 21-24days for Tuberculosis, and the number of mycobacterial colonies counted.

Results

Tuberculosis

Direct Observation Data

Control MDM AAT (5.0 mg/ml)- (no AAT)^(a) exposed MDM Experiment 1 20%4% Experiment 2 17% 6% ^(a)percent of cells infected with M.tuberculosis.

Colony Count Data

In a separate experiment, we cultured the cell-associated TB toindependently confirm the inhibitory AAT effect. The TB counts per mlwere 1.6.times.10.sup.5 per ml in the control MDM cultures and0.57.times.10.sup.5 per ml in the AAT-exposed cultures, an inhibitoryeffect of 64% due to the presence of AAT.

Mycobacterium avium Complex

We used the related mycobacterial organisms known as Mycobacterium aviumcomplex (MAC). MAC is important because it is a leading cause ofinfectious disease in AIDS patients. It is also a difficult problem innormal people who contract this infection; it is very difficult totreat, and sometimes impossible to treat with current antimicrobialdrugs. Using AAT or an AAT-like molecule may represent a novel means oftherapy in these infections.

Direct Observation Data

Control MDM AAT (5.0 mg/ml)- (no AAT)^(a) exposed MDM Experiment 1 17%10% a. ^(a)percent of cells infected with M. tuberculosis.

Colony Count Data

FIG. 1 shows the results of 4 separate experiments that demonstrate thatAAT significantly blocks infection of MDM with MAC with a mean effect ofapproximately 55% inhibition. These experiments were conducted asdescribed above. The AAT mimic refers to ala-ala-pro-val-chloromethylketone (an AAT-like synthetic serine protease inhibitor)(Supplier:Bachem). The AAT mimic results confirm the AAT data using an independentspecies, and provide proof that the concept of serine proteaseinhibition to treat mycobacterial infections extends to small moleculeinhibitors that make attractive drug candidates.

In the same cultures depicted above, we measured the concentration ofthe pro-inflammatory cytokine TNFα. As shown in FIG. 2 and FIG. 3, AATand the AAT mimic both significantly inhibited the production of TNFα inthe MDM cultures by up to 100%. The blockade of pro-inflammatorycytokine production may represent an additional mechanism by whichserine protease inhibitors block infection with TB and with MAC.

Example Two

Clinical Study in MAC Infection

The data described above in vitro using MAC have been supplemented witha clinical study. In this clinical investigation, AAT phenotypes(alternative forms of the AAT protein) were assessed in patients withdocumented lung infection with MAC and who had lung disease. Thesepatients were compared to a control group consisting of patients withthe lung disease bronchiectasis (in order to show that the presence oflung disease alone did not account for the presence of MAC infection).

MAC Infection Bronchiectasis N = 134 subjects (lungs) (lung disease)P-value Sex- Male 8.97% 23.21% Female 91.3% 76.79% Age (mean) 64.5 yrs64.0 yrs AAT phenotype 0.006 (% abnormal)- YES 27.7%  5.3% NO 72.3% 94.7%

Note in this table that that for the control (bronchiectasis) group, theproportion of patients with abnormal AAT molecules is 5.3%. This is inmarked contrast to the case in the MAC-infected group, where theproportion is 27.7%, a 5.2 fold increase. The MAC-infected patients were5.2 times as likely as the control group to harbor an abnormal form ofAAT. This establishes a clinical link between abnormal AAT molecules andinfection with MAC. Thus, the inhibitory role of normal AAT that wediscovered in vitro is borne out in patients.

Example Three

Effect of α-1-Antitrypsin on Stimulated Interleukin-1 Beta Production inWhole Human Blood

Design: Venipuncture was performed on 3 healthy volunteers using a21-gauge needle, and the venous blood was aspirated into a heparinizedtube. Blood was then aliquoted into 6 milliliter polypropylene tubes anddiluted 1:4 with sterile RPMI tissue culture medium alone (Control),diluted 1:4 in medium containing heat-killed Staphylococcus epidermidisat a final concentration of 1:1000 as a stimulus (Staph), or into tubescontaining Staphylococcus epidermidis and α-1-antitrypsin (AAT, Aralast®from Baxter). All cultures were then incubated .times.24 hrs at37.degree. C./5% CO.sub.2). Following incubation, the samples werecentrifuged .times.1,500 g, and the supernatants collected. Supernatantswere assayed for interleukin-1 beta concentration using a validatedelectrochemiluminescence apparatus that quantifies cytokine proteins.

RESULTS: The data are presented as the mean.+−.SEM interleukin-1 betaproduction, and the values are shown on the vertical axis. As shown, AATsignificantly inhibited Staph-stimulated inteleukin-1 beta productiondose-dependently, and the inhibition was observed at all concentrationstested (See FIG. 5).

DISCUSSION: The inventors have shown herein for the first time that AATblocks IL-1 beta production as an example of proinflammatory cytokineproduction. IL-1 beta is crucial for development of the symptoms and/ormanifestations of anthrax disease. The results presented in this examplesupplement the already supposed mechanism by which AAT may be used as atherapeutic agent to cure anthrax by blocking the production of theactive toxin.

Example Four

In outpatient pneumonias, it is known that gram-positive organismspredominate whereas in the intensive care unit (ICU), gram-negativepneumonias are disproportionately incident.

The pathogenesis of pneumonia involves colonization followed bymicro-aspiration. Persons in the ICU become colonized with gram-negativerods. Therefore, it is apparent to physicians that only sick persons inthe ICU become colonized with gram-negative rods. Processed fibronectinis an important receptor for gram-negative bacilli in vivo.

One means of treating patients with gram-negative pneumonias would be toblock gram-negative rod colonization. For example, in health,unprocessed fibronectin is not a receptor for gram negative bacteria.During illness, secretions become rich in serine proteases. Serineproteases process (proteolize) fibronectin. Processed fibronectin is areceptor for gram negative bacteria. This results in colonization. Theuse of serine protease inhibitors like α-1 antitrypsin or any of thefunctional derivatives thereof as disclosed in this application can beused by one of ordinary skill in the art to block gram-negative rodcolonization and therefore treat Gram-negative pneumonias. Thus, serineprotease inhibitors like AAT can be administered topically using topicalformulations including, for example, but not limited to, liquid, cream,aerosol, etc., to block colonization of the epithelium by Gram negativerods. Representative examples of publications providing non-limitingexamples of Gram negative bacilli that may treated using thecompositions of the present invention may be found in Charlotte L.Barey-Morel et al. The Journal of Infectious Diseases VI 155, No. 4(1987); W. G. Johanson et al. Annals of Internal Medicine 77: 701-706(1972); W. G. Johanson et al. The New England Journal of Medicine Vol281 No. 21 (1969); James J. Rahal et al. JAMA Vol. 214 No. 4 (1970), theentire texts of each of which are incorporated by reference.

In a similar fashion serine protease inhibitors like AAT could beadministered topically using topical formulations including, forexample, but not limited to, liquid, cream, aerosol, etc., to treatbacterial infections caused by Gram positive organisms.

Likewise, in a similar fashion serine protease inhibitors like AAT couldbe administered topically using topical formulations including, forexample, but not limited to, liquid, cream, aerosol, etc., to treatbacterial infections caused by mycobacteria. For the proposed mechanismof action for atypical mycobacteria, please refer to Examples 1 and 2supra.

Throughout this application various publications and patents arereferenced. The disclosures of these publications and patents in theirentireties are hereby incorporated by reference into this application inorder to more fully describe the state of the art to which thisinvention pertains.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

What is claimed:
 1. A pharmaceutical compositions comprising: a firstagent comprising a fusion polypeptide comprising, a first polypeptidecomprising mammalian alpha-1 antitrypsin (AAT) and a second polypeptidecomprising an IgG1 Fc; and a second agent comprising at least one of: anantimicrobial agent comprising a macrolide antibiotic agent, anon-macrolide antibiotic agent, other anti-bacterial agent, ananti-fungal agent, an anti-viral agent, an anti-parasitic agent, otherantimicrobial agent; an anti-inflammatory, or an immunomodulatory agent,wherein said AAT is represented by SEQ ID NO: 69 or SEQ ID NO:
 79. 2.The pharmaceutical composition according to claim 1, wherein a macrolideantibiotic agent comprises one or more of a synthetic, semi-synthetic ornaturally occurring macrolide antibiotic.
 3. The pharmaceuticalcomposition according to claim 1, wherein a macrolide antibiotic agentcomprises one or more of methymycin, neomethymycin, YC-17, litorin,erythromycin A to F, oleandomycin, dirithromycin, flurithromycin,davercin, josamycin, kitasamycin, spiramycin, midecamycin, rokitamycin,miokamycin, lankacidin, clofazamine, troleandomycin, isonicotinic acidhydrazide, rifampin and isoniazid, isoniazid, levofloxacin,troveofloxacin, pefloxacin, gatifloxacin, and moxifloxacin, ethambutol,penicillins, cephalosporins, carbacephems, cephamycins, carbapenems,monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines,macrolides, oxazalidinones, fluoroquinolones, penicillin G, methicillin,nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin,amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin,piperacillin, imipenem, aztreonam, cephalothin, cefazolin, cefaclor,cefamandole formate sodium, cefoxitin, cefuroxime, cefonicid,cefinetazole, cefotetan, cefprozil, loracarbef, cefetamet, cefoperazone,cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefepimem, cefixime,cefpodoxime, cefsulodin, fleroxacin, nalidixic acid, norfloxacin,ciprofloxacin, ofloxacin, enoxacin, lomefloxacin, cinoxacin,doxycycline, minocycline, tetracyclin, amikacin, gentamicin, kanamycin,netilmicin, tobramycin, streptomycin, azithromycin, clarithromycin,erythromycin, erythromycin estolate, erythromycin ethyl succinate,erythromycin glucoheptonate, erythromycin lactobionate, erythromycinstearate, vancomycin, teicoplanin, chloramphenicol, clindamycin,trimethoprim, sulfamethoxazole, nitrofurantoin, rifampin, mupirocin,metronidazole, cephalexin, roxithromycin, co-amoxiclavuanate;Piperacillin and Tazobactam; and salts, acids, bases, and otherderivatives thereof.
 4. The pharmaceutical composition according toclaim 1, wherein the anti-viral agent comprises one or more ofcaspofungin, terbinafine hydrochloride, nystatin, amphotericin B,griseofulvin, ketoconazole, miconazole nitrate, flucytosine,fluconazole, itraconazole, clotrimazole, benzoic acid, salicylic acid,and selenium sulfide or derivative thereof.
 5. The pharmaceuticalcomposition according to claim 1, wherein the anti-parasitic agentcomprises one or more of pirethrins or piperonyl butoxide, permethrin,iodoquinol, metronidazole, diethylcarbamazine citrate, piperazine,pyrantel pamoate, mebendazole, thiabendazole, praziquantel, albendazole,proguanil, quinidine gluconate, quinine sulfate, chloroquine phosphate,mefloquine hydrochloride, primaquine phosphate, atovaquone,co-trimoxazole (sulfamethoxazole/trimethoprim), and pentamidineisethionate or derivative thereof.
 6. The pharmaceutical compositionaccording to claim 1, wherein the anti-fungal agent comprises one ormore of caspofungin, terbinafine hydrochloride, nystatin, amphotericinB, griseofulvin, ketoconazole, miconazole nitrate, flucytosine,fluconazole, itraconazole, clotrimazole, benzoic acid, salicylic acid,and selenium sulfide or derivative thereof.
 7. The pharmaceuticalcomposition according to claim 1, wherein the first polypeptidecomprising mammalian alpha-1 antitrypsin (AAT), comprises mammalian AATrepresented by SEQ ID NO:69.
 8. The pharmaceutical composition accordingto claim 1, wherein the second polypeptide comprises an IgG1 Fcrepresented by SEQ ID NO:65.
 9. The pharmaceutical composition accordingto claim 1, wherein the first polypeptide comprising mammalian alpha-1antitrypsin (AAT), comprises mammalian AAT represented by SEQ ID NO:79.10. The pharmaceutical composition according to claim 1, wherein thecomposition is formulated for delivery by inhalation, oraladministration, intranasal administration, intravenous administration orintra-rectal administration.
 11. A kit comprising: one or morecontainers; and at least one composition comprising a pharmaceuticalcomposition according to claim 1.